MICROBIOLOGICAL REVIEWS, June 1980, p. 331-384 Vol. 44, No. 2 0146-0749/80/02-0331/54$02.00/0 Biology, Immunology, and Cariogenicity of mutanst SHIGEYUKI HAMADAt AND HUTTON D. SLADE* Department of Oral Biology, School ofDentistry, University of Colorado Health Sciences Center, Denver, Colorado 80262 INTRODUCTION 332 ORAL MICROBIAL FLORA 332 ISOLATION AND IDENTIFICATION OF S. MUTAINS AND OTHER ORAL STREPTOCOCCI ...... 333 Characteristic Properties of Oral Streptococci ... 333 S. mutans 333 S. sanguis 334 S. mitior 334 S. salivarius ...... 335 S. milleri ...... 335 Selective Isolation of S. mutans ...... 335 CLASSIFICATION OF S. MUTANS 335 Immunological Typing of S. mutans 335 Serotype-Specific Antigens of S. mutans .... 336 Reactivity of S. mutans with Lectins ...... 340 Cell Wall Structure of S. mutans and Other Streptococci ...... 340 POLYMER SYNTHESIS BY S. MUTANS 342 Extracellular ...... 342 Glucans ...... 342 Fructans ...... 344 -Synthesizing Enzymes ...... 344 Intracellular Polysaccharides ...... 345 Lipoteichoic Acid ...... 345 Interaction of Glucosyltransferase with Various Agents , 346 Invertase 347 a(1-- 6) Glucanase ...... 348 SUGAR BY S. MUTANS ...... 348 ADHERENCE OF S. MUTANS 348 Initial Attachment of S. mutans to Smooth Surfaces ...... 348 Interaction of Salivary Components with Streptococcal Cells ...... 349 Implantation of S. mutans , 350 -Dependent In Vivo Adherence of S. mutans ...... 350 Sucrose-Dependent In Vitro Adherence of S. mutans ...... 350 Cell-to-Cell Adherence: Bacterial Aggregation ...... 352 GENETIC ASPECTS OF S. MUTANS 353 Lysogenicity and Plasmids ...... 353 Transformation 354 BACTERIOCINS OF S. MUTANS: MUTACINS ...... ,., 354 Bacteriocinogeny Among S. mutans . .354 Extracellular Mutacins 355 IMMUNOLOGICAL ASPECTS OF S. MUTANS ...... 356 Distribution of S. mutans Serotypes in ,, 356 In Vitro Effects of Antisera Against S. mutans 356 Immunological Responses of Host to S. mutans ...... 356 Possible Vaccination with S. mutans Antigens ...... 357 CARIOGENICITY OF S. MUTANS IN EXPERIMENTAL ANIMALS ...... 358 Caries Induction in Animals ...... 358 Noncariogenic and Supercariogenic Mutants of S. mutans ,.. 359 S. MUTANS AND DENTAL CARIES IN HUMANS ...... 360 Effect of Sucrose on the Proportion of S. mutans ...... 360 Epidemiological Relationship Between S. mutans and Caries Development .. 360 t The survey of literature pertaining to this review was : Present address: Department of Oral Biology, Osaka Uni- terminated in October 1979. versity Dental School, Nakanoshima, Kita-ku, Osaka, 530 Japan. 331 332 HAMADA AND SLADE MICROBIOL. REV.

PREVENTION OF CARIES CAUSED BY S. MUTANS ...... 361 Suppression of S. mutans by Antimicrobial Agents .361 Inhibition of Adherence of S. mutans by Glucan-Hydrolyzing Enzyes .... 361 ENDOCARDMS CAUSED BY S. MUTANS ...... 362

SUMMARY ...... 363

LITERATlURE CmD ...... 364

INTRODUCTION current state of knowledge concerning S. mu- Whether there is any one bacterium which tans. Numerous reviews and books have re- may always be found in decayed dentine, and cently appeared on microbiological or immuno- which might therefore be entitled to the name logical aspects of dental caries, or oral strepto- of the bacterium of , or whether cocci (27, 46, 196, 265, 303, 397, 438, 445, 494, there are various kinds which occur with consid- 549). erable constancy, we are not able to say. It is ORAL MICROBIAL FLORA now apparent, however, that various microor- ganisms are essential in the pathogenesis of den- The oral microflora is a complex ecosystem tal caries. Orland (451) first demonstrated that which contains a wide variety of microbial spe- selected streptococcal species, namely, entero- cies (Table 1). The mouth is colonized by various cocci, produced dental caries in germfree rats microorganisms before teeth erupt, although when fed a high-sucrose diet. Furthermore, in- newborn infants are essentially free from micro- direct evidence that antibiotics suppressed ex- organisms (394). With the eruption of teeth, perimental dental caries in rodents (396) , distinctive patches primarily of strongly suggested the involvement of certain microbial origin, develop on exposed enamel sur- penicillin-susceptible in dental caries. faces which are covered by a pellicle that is an Since that time, various investigations have amorphous, almost invisible film composed pri- been carried out to elucidate the causative rela- marily of salivary glycoprotein (162, 194). Large tionship between specific oral bacterial species microbial masses develop on the teeth surfaces and dental caries. In 1960, some streptococcal unless proper measures are taken, strains, isolated from carious lesions of rats and hamsters, produced dental caries in "caries-re- TABLE 1. Distribution of bacteria on various sites sistant" rats and hamsters, respectively (157, in the moutha 302). Site Using a sev- fluorescent-antibody technique, Bacterial group Gingi- eral streptococcal strains which shared immu- Plaque Tongue Saliva val nological specificity with the cariogenic strepto- crevice cocci derived from rats and hamsters were iso- Gram-positive facultative 28.2 44.8 46.2 28.8 lated from human carious lesions (275, 628, 629). cocci These strains also produced severe dental caries Streptococci 27.9 38.3 41.0 27.1 in germfree animals. Since then, similar strep- S. mutans (0-50) (0-1) (0-1) (0-30) S. sanguis (40-60) (10-20) (10-30) (10-20) tococcal species have been isolated from human S. mitior (20-40) (10-30) (30-50) (10-30) carious lesions by several investigators (183, 202, S. salivarius (0-1) (40-60) (40-60) (0-1) 321, 322). Carlsson (54, 55) indicated that prop- S. milleri (3-25) (0-1) (0-1) (14-56) erties of these cariogenic streptococci were sim- Staphylococci 0.3 6.5 4.0 1.7 Gram-positive anaerobic 12.6 4.2 13.0 7.4 ilar to those originally isolated from human car- cocci ious teeth by J. K. Clarke (81) in 1924 to which Gram-negative anaerobic 6.4 16.0 15.9 10.7 he had given the species name mutans. Thus, cocci the rediscovery of Streptococcus mutans fol- Gram-negative faculta- 0.4 3.4 1.2 I 0.4 tive cocci lowed the original observation by about 36 years. Gram-positive facultative 23.8 13.0 11.8 15.3 S. mutans is now considered to play an im- rods portant role in the development of dental caries Gram-positive anaerobic 18.4 8.2 4.8 20.2 in animals and humans. Extensive research on rods Gram-negative faculta- NDb 3.2 2.3 1.2 this microorganism has been done during the tive rods last 10 years. Unfortunately, however, S. mutans Gram-negative anaerobic 10.4 8.2 4.8 16.1 is not given an independent species position in rod the newest edition of Bergey's Manual ofDeter- Spirochetes ND ND ND 1.0 minative Bacteriology (43). It will be recognized Modified from Gibbons and van Houte (194, 196) and from the evidence described below that S. mu- Mejare and Edwardson (403). Data are expressed as a per- tans is the best-defined centage of total cultivable count on anaerobically incubated species among the oral blood agar. Data in parentheses are expressed as a percentage streptococci. of the total facultative streptococcal counts. The present review is an attempt to define the h ND, Not detected. VOL. 44, 1980 STREPTOCOCCUS MUTANS 333 whereas desquamation of epithelial cells does problems in obtaining representative samples not permit the heavy accumulation on oral mu- from different oral sites, and in dispersing, cul- cosal surfaces such as the dorsum of the tongue tivating, and enumerating the microorganisms. (195). The number of bacteria in dental plaque No single cultivation method of examining the can reach 108 per mg (wet weight) (192). complex and variable dental plaque flora will As shown in Table 1, predominant microbial satisfy all the necessary conditions. Strictly an- species are significantly different in different aerobic procedures will be required in many sites. Irrespective of variation from sample to cases. It is fortunate, however, that most oral sample, streptococci, gram-positive rods, and streptococcal species can be isolated from var- veillonellae comprise the majority of the total ious sites in the mouth by using a selective viable count. In plaque and gingival crevice, medium, mitis salivarius (MS) agar (Difco Lab- higher proportions of gram-positive and -nega- oratories, Detroit, Mich.). Although MS agar tive rods are observed. More recently, it has was originally devised by Chapman to isolate been demonstrated that samples obtained from fecal streptococci, the use of MS agar has dom- deep periodontal pockets in patients with ad- inated other cultural methods for the isolation vanced periodontitis and periodontosis consist of oral streptococci, including S. mutans, be- of a significantly higher percentage ofgram-neg- cause of its selective and differential properties. ative anaerobic rods (440, 536, 537). The increasing attention associated with the Clinical observations in humans and animals occurrence ofS. mutans in the various lesions of indicate that plaque formation is an essential the human teeth has resulted in a more refined requirement for both dental caries and periodon- methodology regarding its isolation, quantita- tal disease. It is of interest to note that a limited tion, and species identification. number of bacterial species in the oral flora can On MS agar medium, most oral streptococci be detected on the tooth surface. Ritz (472) show a characteristic colonial morphology which described the shift in microbial population in permits their provisional differentiation. Usu- developing plaque from a preponderance of coc- ally, the agar plate is incubated in an atmosphere cal forms in very early plaque with an increase of 95% nitrogen and 5% carbon dioxide at 37°C of rods and filament forms with age. However, for 1 to 2 days, followed by incubation in air for streptococci make up the greater number of the another 1 to 2 days. A candle jar or the GasPak total bacterial population in plaque throughout system (BBL Microbiology Systems, Cockeys- the period. Most of the streptococci can be iden- ville, Md.) can also be used for primary isolation tified as one of the following species: S. mutans, of oral streptococci from clinical samples. S. sanguis, S. mitior, S. salivarius, and S. milleri In addition to the characteristic colonial mor- (52, 54, 55, 126, 202, 245, 403). phology, oral streptococci can be differentiated It appears that certain oral streptococcal spe- by their ability to ferment certain sugars (espe- cies have a predilection for colonizing particular cially mannitol and ) and to adhere to sites in the mouth. S. sanguis and S. mutans smooth surfaces in the presence of sucrose (245). preferentially colonize the surfaces Techniques for these tests are described in detail and prosthetic devices (54, 59, 62). S. salivarius (92). is present in low numbers in plaque, whereas there is no preferred site for S. mitior in the oral Characteristic Properties of Oral cavity. Whereas S. salivarius is an early colo- Streptococci nizer in the mouth after birth, S. sanguis is not S. mutans. S. mutans was isolated from hu- usually found until the teeth erupt (59, 60). man carious lesions by Clarke (81) in 1924. His Similar findings have been obtained with S. mu- is as follows: tans. The preferred habitat ofS. mutans appears description to be tooth surfaces. The numbers of S. sanguis S. mutans was isolated from 36 of the 50 teeth. Acid is very isolated from previously cleaned teeth were rapidly produced, the medium, originally pH 7, giving a reac- much higher than those of S. salivarius, indi- tion of pH 4.2 in about 24 hours. All the strains isolated of ferment , , raffinose, mannite (mannitol), inulin, cating the importance of the selective ability and salicin with production of acid. There is usually neither streptococci to attach to oral surfaces. The ob- haemolysis nor discoloration on blood-agar. The fact that the served affinity ofthese species for an oral surface colonies of S. mutans adhere closely to the surface of the teeth is reported to correlate positively with the pro- appears to be of great importance. portions that are found in vivo (195). The occurrence ofS. mutans in human carious lesions was confirmed (108, 183, 220, 321, 382). ISOLATION AND IDENTIFICATION OF Extensive taxonomic studies revealed that these S. MUTANS AND OTHER ORAL organisms formed a fairly homogeneous group STREPTOCOCCI of nonmotile, -negative, gram-positive In general, there are many difficult technical streptococci (55, 126, 143, 202). A number of 334 HAMADA AND SLADE MICROBIOL. REV. investigators have also revealed an association subacute endocarditis, that split arginine and between the occurrence of S. mutans and the esculin and produce glucan from sucrose. They development of caries (vide infra). produce hydrogen peroxide when grown aerobi- As tabulated in Table 2, most streptococcal cally. Carlsson (52) demonstrated that the main strains that ferment mannitol and sorbitol in habitat of S. sanguis in humans is the oral addition to various other sugars, and synthesize cavity, especially in plaque. Low levels of S. adherent water-soluble glucan from sucrose, are sanguis were reported in human feces (595). considered S. mutans. They do not usually On MS agar, S. sanguis produces small zoo- deaminate arginine to produce ammonia. S. mu- gleic colonies with a firm consistency which are tans is mostly a- or y-hemolytic on sheep blood embedded in the medium and which deform the agar, but a few /i-hemolytic strains (621) have surrounding agar. Many S. sanguis strains pro- been reported. A further characterization of duce spreading zones typical of twitching motil- these ,B-hemolytic strains is needed before they ity on blood agar plates (252). can be identified as S. mutans. S. mutans has Strains with a similar colonial morphology been subclassified into several types based on which do not hydrolyze arginine and esculin but immunological, biological and genetic proper- synthesize glucan are considered to be another ties. These properties will be discussed below in type of S. sanguis (55). Although other investi- detail. gators considered the latter strains as glucan- The natural habitat ofS. mutans is the human producing S. mitior (91, 97, 98), they are in- mouth. The organism can be isolated frequently cluded within the species sanguis for conveni- from feces in humans (149, 307, 550) and rats ence and are separated into biotypes A and B as (261, 576). Although S. mutans appears not to shown in Table 2 (144, 579). be widely distributed in wild animals, Dent et Serological studies on S. sanguis strains dem- al. (104) isolated S. mutans from the Patas mon- onstrate the presence of at least three (488) or key and Indian fruit bat of 18 animal species four (579) types. The close relationship of S. examined. Coykendall et al. (96) and Lehner et sanguis to group H streptococci has been sus- al. (343) also isolated S. mutans from wild rats pected for many years, but still remains to be inhabiting sugar cane fields and from rhesus defined (88, 144, 146, 245, 252, 490). In spite of monkeys. It has also been isolated from experi- the complexity of its antigenic structure, S. san- mental rats and hamsters (174). guis is not difficult to identify because of the To compare and differentiate other oral strep- unique physiological properties and colonial tococcal species from S. mutans, the following morphology on sucrose agar. brief summary of the other principal species is S. mitior. S. mitior, frequently called S. mitis, given. is an a-hemolytic, bile-sensitive streptococcus S. sanguis. The species name was given by that does not hydrolyze arginine and esculin. It White and Niven (614) to the a-hemolytic strep- is peroxidogenic, but does not ferment inulin, tococci, isolated from the blood of patients with sorbitol, and mannitol. On MS agar, it elaborates

TABLE 2. Generalized key characteristics for identifying the predominant streptococcal speciesa Fermentation Hydrolysis Hemol- ysis on Polysaccharide from Perox- sheep Organism Man- Sorbi- Meli- Raffi- Escu- In1 Argi- Escu- sucrose ide blood nitol tot biose nose lin nuli nine lin agar plate S. mutansb a + + + + + + - + Glucan >>fructan + 8 b + + + + + + + + Glucan >> fructan - -y c/elf + + + + + + - + Glucan >>fructan - y dlg + ± - - - + - + Glucan >> fructan + 8 S. sanguis' A - - - + + + + + Glucan + a B ------Glucan + a S. mitior - - - ± - - - +- + a S. salivarius - - - + + + - - Fructan >> glucan - y S. milleri - - - - + - + + - - a/y a Collected data from references 55, 91, 144, 202, 221, 245, 403, 459, and 522. b Serotypes according to Bratthall (31) and Perch et al. (459). c Biotypes according to Torii (579). VOL. 44, 1980 STREPTOCOCCUS MUTANS 335 soft, round, black-brown colonies. Some "S. mi- completely inhibited growth of serotype a tior" strains produce extracellular glucan from strains (349, 545). sucrose, and develop colonies that are indistin- More recently, Linke (353) devised a new se- guishable from those of S. sanguis. Deoxyribo- lective MSFA medium for S. mutans which in- nucleic acid (DNA) base sequence studies cludes mannitol, sorbitol, basic fuchsin, and so- showed the presence of two types of moderately dium azide. On the other hand, BCY (267) and homologous strains (97). Perhaps "glucan-pro- MMIO sucrose agars (369) are nonselective me- ducing S. mitior" can be included in the biotype dia which allow total bacterial counts and B S. sanguis (Table 2), or perhaps these strains enumeration of polysaccharide-synthesizing should be given a new taxonomic designation streptococci, including S. mutans. (144). Comparative evaluation of various selective S. salivarius. S. salivarius primarily pro- media under standardized conditions indicates duces levan (fructan) from sucrose, and there- that the growth of most serotypes of S. mutans fore forms unique large, domed colonies on MS is depressed by the use of such media. The agar. It does not ferment sorbitol and mannitol. variable cultural results prevent the unqualified Colonies of S. salivarius are nonhemolytic on use of a single medium for isolation and enu- blood agar and are not peroxidogenic. The meration of S. mutans (133, 360). tongue is its main habitat in the oral cavity (55, 91). S. milleri The species name milleri was orig- CLASSIFICATION OF S. MUTANS inally proposed by Guthof (214) for streptococci Immunological Typing of S. mutans which had been isolated from dental abscesses. As described in the preceding section, isola- They deaminate arginine, do not ferment man- tion and identification have relied primarily on nitol and sorbitol, do not produce extracellular phenotypical characteristics rather than immu- polysaccharides from sucrose, and do not pro- nological specificity. Strains of S. mutans are duce peroxides. They can be isolated from the phenotypically homogeneous, as confirmed by gingival crevice and dental plaque (Table 1). S. numerical taxonomic studies (55, 116). However, milleri shows some resistance to sulfonamides recent investigations have revealed a great de- and (403), and therefore can be grown gree of heterogeneity of S. mutans when sur- on the selective medium devised for isolation of veyed serologically, genetically, and biochemi- S. mutans (see Selective Isolation of S. mutans). cally. Although S. milleri constitutes a fairly homo- Zinner et al. (628) first demonstrated a sero- geneous group based on its cultural and bio- logical heterogeneity in S. mutans strains FAl chemical characteristics, it appears that immu- and HS1, which were of rat and hamster origin, nological specificity is heterogeneous. respectively. In a later study, Bratthall (30, 31) described the presence of five serotypes, a, b, c, Selective Isolation of S. mutans d, and e, within the species. Subsequently, Perch MS agar is most widely used to isolate S. et al. (459) revealed two additional serotypes, f mutans as well as other oral streptococcal spe- and g. The serological techniques used in these cies. Although MS agar is available commer- studies were the capillary precipitin test, origi- cially, recent investigations (349, 545) have dem- nally introduced by Lancefield (334) in classify- onstrated significant discrepancies between data ing beta-hemolytic streptococci; immunodiffu- from different MS agar preparations manufac- sion; comparative immunoelectrophoresis; and tured by different companies for the recovery immunofluorescence. The specific antigens of and quantitative enumeration offreshly isolated each serotype have been purified and character- and stock strains of S. mutans. ized chemically (see Serotype-Specific Antigens Linke (353) indicated that trypan blue in MS of S. mutans). agar will inhibit growth of most S. mutans Coykendall (94) has reported analysis of DNA strains. Furthermore, addition of Chapman tel- base composition and DNA base sequence sim- lurite solution to MS agar resulted in a signifi- ilarities of S. mutans strains, showing the pres- cant reduction in the number of colonies (360). ence of four genetic groups (I through IV), or MS agar has been modified to be more selec- "genospecies." These four groups correlated tive for the isolation of S. mutans by adding with four serotypes, c, b, a, and d, respectively. either sulfonamide (MC agar; 53), bacitracin More recently, he (95) proposed to give the (MSB agar; 198), polymyxin (154), or even sup- subspecies of S. mutans species names (Table plemental sucrose (MS40S agar; 266). However, 3). A considerable overlap in the mole percent it has been suggested that some serotype d/g guanine plus cytosine content of these species is strains are susceptible to sulfonamide (133, 360), apparent. and incorporation of bacitracin into MS agar Heterogeneity has also been observed in var- 336 HAMADA AND SLADE MICROBIOL. REV. TABLE 3. Chemical composition and immunological determinant oftype-specific antigen preparations from S. mutans Composition (wt %) Sero- S Extraction Proposed an- Refer- type Stram Source procedure Galac- Glu- Rham- Galac- Phos- tigenic deter- ence tose cose nose tmmne phorus a HS6 Cells, Boiling 54 10 - 5 5.0 0.3 Glc-,B(1,6)- 426 walls water Glc b FAl Cells, Cold 27 - 47 2 5.4 2.3 a-Gal 425 walls TCAb c Ingbritt Cells Cold TCA - 29 69 - 0.5 0.5 Glc-a(1,4)- 355 Glc c GS5 Walls Hot form- - 29 43 - NDc ND Glc-a(1,4)- 612 amide Glc d B13 Cells, Cold TCA 62 33 - - 1.6 0.3 Gal-,B(1,6)- 357 walls Glc e MT703 Cells Hot saline - 37 56 - 5.0 0.3 Glc-fi(1,6)- 231 Glc e V-100 Walls Hot form- - 24 52 - ND Trace Glc-fl(1,4)- 613 amide Glc f OMZ175 Celis, Hot TCA - 47 49 - Trace 0.2 Glc-a(1,6)- 216 walls Glc f MT557 Cells Hot saline - 39 59 - Trace 0.2 Glc-a(1,6)- 216 Glc g 6715 Cells Hot buffer 61 10 - - 9.5 0.4 ,B-Gal 264 a Glc, Glucose; Gal, galactose. b TCA, Trichloroacetic acid. 'ND, Not described. ious enzyme proteins, such as dehydrogenases isolates (221) belonging to serotype d or g pro- (39,40), glucosyltransferase (75), aldolases (375), duced a markedly zoogleal colony on MS agar, and invertases (385, 565) within the species S. exhibited alpha- on sheep blood agar, mutans. and were peroxidogenic, whereas those belong- In contrast, Shklair and Keene (521) proposed ing to serotype c, e, or fdeveloped a small, rough, a biochemical scheme for the separation of S. raised and undulated colony as shown in Fig. 1 mutans into five biotypes, which they reported and were nonhemolytic and nonperoxidogenic to correlate with serotypes a to e. The biotyping (Table 2). Moreover, 23 isolates of serotypes d was based on the fermentation ofmannitol (with and g were strongly agglutinated shortly after or without bacitracin), sorbitol, raffinose and addition of T2000 (500 ,tg/ml, final con- melibiose and the production of ammonia from centration), and 40 out of 41 serotype c/elf arginine. They later refined their scheme to in- strains were not agglutinated even after 18 h of clude additional serotypes f and g and desig- incubation. Similar results have been obtained nated them biotypes I to V (522) (see Table 3). with reference strains of S. mutans (622). These It is of interest to note that bacitracin inhibited results indicate that serotypes d and g and se- acid production by serotype a (biotype III) rotypes c, e, and f constitute two major sub- strains but not serotype c, e, and f strains (bio- groups, which correlate with the genospecies type I) that were otherwise similar to biotype III proposed by Coykendall (95). (Table 2). In summary, to minimize taxonomical confu- However, a study of 137 clinical isolates com- sion of S. mutans, we prefer serotypes to geno- posed of serotypes c or e (221) indicates that species or biotypes. Serotyping is a routine pro- serotypes c and e are essentially similar with cedure and is as valuable as that used with other respect to fermentation ofmelibiose, a key char- streptococcal immunological groups and types. acter in the differentiation of biotypes I and V. Use ofthis scheme for differentiation ofserotype Serotype-Specific Antigens of S. mutanm c and serotype e S. mutans would result in an The antigenic components of S. mutans can error in the count of serotype e S. mutans. be extracted in a soluble form by various meth- Therefore, it appears that biotyping is not cor- ods from whole cells or cell walls. These methods related with serotyping, and the suitability of involve the use of hot physiological saline (50, biotyping as a taxonomical tool remains in 216, 220, 230, 231, 234), 5 to 10% trichloroacetic doubt. acid (216, 356, 357, 425, 529, 586, 601), formamide It should be added here that all the clinical (39, 172, 612), dilute hydrochloric acid (31, 334), VOL. 44, 1980 STREPTOCOCCUS MUTANS 337

IA

FIG. 1. Typical colonial morphologies of S. mutans on MS agar. Colonies of serotype d and g strains are surrounded by a puddle (zooglea) with a gelatinous consistency (top), whereas those of serotype c, e, and f strains are small, raised, irregular in margin, and adherent, but do not show the zooglea (bottom). (Reproduced with permission, reference 221.) 338 HAMADA AND SLADE MICROBIOL. REV. phosphate buffer, pH 7.3 (264), cell wall lytic S. mutans serotypes have been reported to con- enzyme "mutanolysin" (230), or even water tain a significant quantity of rhamnose (22, 244), (426). The crude antigen extracts usually contain the purified antigens do not contain rhamnose contaminants such as nucleic acids, unidentified (Table 3). Considerable cross-reactivity has been cellular proteins, and cross-reacting polyglycer- observed among serotypes a, d, and g (31, 219, ophosphate (PGP) antigens as well as the type- 356, 459). specific antigens (234). Therefore, serotype-spe- A rhamnose-rich polysaccharide has recently cific antigens ofS. mutans have been purified by been isolated from cell walls of strain B13, se- a variety of column chromatographic proce- rotype d (464). It is composed of rhamnose and dures, including gel filtration, ion-exchange glucose, whereas the serotype antigen from this chromatography, and affinity chromatography. strain is a galactose/glucose polymer (357). It is The purified antigens from serotype a to g S. immunologically distinct from the latter. Other mutans strains are polysaccharides located in or distinct antigenic polysaccharides will probably on the cell wall (Table 3) (354, 531). They are be found in the cell walls of S. mutans. composed primarily of a combination of either The earlier literature reflects confusion in the glucose, galactose, or rhamnose. They differ serotyping of some S. mutans strains (31). For from the group-specific polysaccharides of cer- example, strain 6715 was originally reported as tain 8l-hemolytic streptococci (326) in the ab- serotype d (264), but was later reclassified as sence of significant quantities of N-acetylgluco- serotype g, using appropriately absorbed typing samine and N-acetylgalactosamine. A small serum (219). Furthermore, although strain AHT quantity of these amino sugars is present in the was originally reported to be serotype a, it has a and b antigen. Table 3 summarizes the com- been found that some AHT substrains, including position of the seven polysaccharides and the the one deposited in the National Collection of probable antigenic determinant of each. The Type Cultures, London, are serotype g and not chemical composition and recovery of these a (219; R. R. B. Russell, personal communica- polysaccharides (Table 3) illustrate a variation tion). in their state of purity. This is due in large Quantitative precipitin inhibition tests indi- measure to the source of the polymer and to the cate that the serotype a, c, e, and f antigenic various methods of extraction and purification. determinants depend mainly upon a glucose-glu- However, reasonably good agreement has been cose sequence (216, 231, 355, 426, 612, 613). The obtained in the gross composition of the antigen specificity appears to be related to the presence in each of the two strains of types c, e, and f. of either a and b forms and (1 -- 4) and (1 -+ 6) Confirmation of the immunological determinant linkages (Table 3). On the other hand, the se- must await studies on more highly purified ma- rotype d and g specificities depend on a config- terial. uration of galactose and glucose within the an- The a (strain HS6) (426), d (strain B13) (357), tigen molecules (264, 357). However, another and g (strain 6715) (219, 263, 264) serotype poly- group (42) reports that the serotype a antigenic saccharides are composed principally of glucose determinant may be D-galactose rather than D- and galactose. Although the cell walls of these glucose and that the d specificity depends upon a terminal D-glucose. The result of Brown and TABLE 4. Taxonomic relationship between Bleiweis (42) confirms the D-galactose specificity serotypes and biotypes ofS. mutans andproposed and the a-d antigenic sites as reported by Mu- genospecies kasa and Slade (426) and Linzer and Slade (357). S. mutans DNA base Serotype c (strains Ingbritt and GS5) (355, Proposed species content 612), e (strains MT703 and V-100) (231, 613), a ~~~namec (MOl% Serotypea Biotype' G+C)d and f (strains OMZ175 and MT557) (216) anti- genic polysaccharides are essentially composed c, e, f I S. mutans 36-38 b II S. rattus 41-43 of glucose and rhamnose. Hapten inhibition a III S. cricetus 4244 studies suggest that an a-glucosyl residue is the d, g IV S. sobrinus 44-46 immunological determinant of the serotype c c _ S. ferus 4345 and f antigens, whereas a f-glucose residue is e V the serotype e determinant (216, 355, 612, 613). a From Bratthall (31) and Perch et al. (459). The type f antigen from strain OMZ175 is b From Shklair and Keene (522). related to dextran. This is indicated by the pres- c From Coykendall (95). ence of a-1,6-glucosidic linkages (90% inhibition d From Coykendall (95). G+C, Guanine plus cyto- of the precipitin reaction by isomaltose and a- sine. methyl-D-glucopyranoside), adsorption to and e, Not described. release from a concanavalin A (ConA)-Sepha- VOL. 44, 1980 STREPTOCOCCUS MUTANS 339 rose column, and reaction with antidextran se- antigen can be extracted by 0.1 M NaOH at rum (216). 60°C for 30 min. The b antigen as well as the This is the only S. mutans type antigen which PGP/lipoteichoic acid (LTA) antigen (vide in- reacts with ConA and dextran antiserum. The fra) appear to be destroyed by this procedure. cross-reaction between type a and d strains is In this context, the electrophoretic mobility of due to a common antigenic site (a-d) which is 16 strains of S. mutans was compared by a the same in both cases. The two antigenic spec- microelectrophoresis technique, and it was ificities (a and a-d; d and a-d) are present on a found that two serotype b strains examined single polysaccharide molecule in each case showed the highest surface potential (448). This (356). This specificity is due mainly to a-D-ga- may be due to the protein content of the two lactose-1 -- 6-glucose (Table 3). The D-galactose antigenic forms of the type b antigen (425). One specificity of the a-d site has recently been con- form contains 30% protein, in addition to poly- firmed (42). saccharide. Many of the amino acids in this Serotype e antigen cross-reacts with Lance- protein were those not present in the peptido- field group E antiserum (31). However, this an- glycan of S. mutans (534). The antigenic speci- tigen has two different immunological specifici- ficity was released from cell walls with lysozyme, ties. One shares a specificity with group E strep- although the walls were not dissolved. Also, tococci (543), and the other is specific for sero- trypsin and pepsin did not release it (426a). type e S. mutans (230, 231). The immunological Consequently, the protein bound to the immu- distinction, based upon a single antigen mole- nologically specific carbohydrate is not the pep- cule, differs from the original description of the tide of the cell wall peptidoglycan but another antigen (31). We use antiserum against the peptide/protein located in the wall. whole cells of serotype e S. mutans rather than As discussed above, immunological cross-re- antiserum against Lancefield group E cells, be- actions are frequently observed among some cause the former serum possesses far more in- combinations of serotypes, i.e., serotypes c, e, tensive immunological reactivity with serotype and f and serotypes a, d, and g. These close e S. mutans (220, 230, 231). relationships correlate with the DNA base se- A cross-reaction between serotype c antiserum quence data (195) and the biochemical separa- and one of the serotype e antigens has been tion data (221, 459, 522). These cross-reactions reported (613). The cross-reacting antigen may are evident by immunodiffusion, whole cell or be a degraded product resulting from drastic cell wall agglutination, and immunofluorescence formamide extraction, thus exposing internal a- (31, 32, 37, 219, 220, 244, 356, 402). Serotype glucosidic linkages which may be reactive with specificity was obtained after appropriate ad- serotype c antiserum. Some other aspects of sorption procedures. cross-reactive phenomena will be discussed in It has also been shown that many gram-posi- the next section. tive bacteria, including all strains of S. mutans, The nature of the serotype b antigen has not possess a common antigenic component, PGP been adequately explained. Mukasa and Slade (72, 90, 234, 312, 313, 395, 426, 617). Crude anti- (425) obtained two forms of polysaccharide an- gen extracts of S. mutans serotypes a to g re- tigen by chromatographic purification from acted with anti-PGP serum in agar gel. The strain FAl. The chemical composition and elec- cross-reactive PGP antigen was adsorbed with trophoretic mobility ofthe two forms are consid- an anion-exchange resin (234). Many batches of erably different; however, they possess an iden- antiserum against S. mutans whole cells react tical immunodeterminant. These antigens con- with heterologous antigen extracts of various tain low amounts of phosphorus and glycerol, serotypes and species of streptococci when ex- but they are negatively charged. They are ad- amined by the passive hemagglutination tech- sorbed to diethylaminoethyl-Sephadex A-25 nique (223). resin (234). On the other hand, Vaught and Recently, it was found that antiserum specific Bleiweis (601) purified two antigenic compo- for serotype e S. mutans glycosyltransferase nents from another serotype b strain, BHT. One (GTase) almost completely inhibited the GTase antigen appears to be identical to one of the activity of types c, e, and f S. mutans, whereas polysaccharide b antigens of Mukasa and Slade the GTase of types a, d, and g was not affected (425), but the second antigenic component, by the antibody (236). On the other hand, it has which is more negatively charged, is reported to been reported that antiserum against serotype a be a glycerol teichoic acid substituted with a S. mutans GTase reacted with GTase of type a, galactosyl moiety. Hapten inhibition studies d, and g strains, but not with those of type b and suggest that the immunological determinant of c strains (171). Several other reports (139, 175, serotype b is a f)-D-galactoside (425, 601). Fur- 229, 332, 538) also indicate that serotypes c, e, thermore, all serotype a to g antigens except b and f and types a, d, and g S. mutans can be 340 HAMADA AND SLADE MICROBIOL. REV. separated into two major groups on the basis of hibited the in vitro adherence of S. mutans 6715 the immunological relatedness of GTase protein (serotype g) to a glass surface. In this case, the molecules. lectin may have bound to the polysaccharide In contrast, an antiserum against an S. mutans component of GTase. type a (HS6) purified GTase, which produced 95% water-insoluble glucan, inhibited the Cell Wall Structure of S. mutans and GTases from serotypes a, b, c, and d (358). Other Streptococci Antisera against similar purified GTase prepa- The streptococcal cell wall contains four ma- rations from other type a strains need to be jor antigenic polymers: peptidoglycan, group- tested to clarify these results. and type-specific polysaccharides, protein, and In this connection, Russell (495) has shown on the glycerol form of teichoic and lipoteichoic the basis of sodium dodecyl sulfate-gel electro- acids. A considerable volume of evidence indi- phoresis of whole cell proteins that strains of cates, in contrast to a layer type structure (326, serotypes a and b have unique patterns. Sero- 326a), the existence of a mosaic structure in type d and g strains show a very close relation- which each of these polymers is accessible for ship to each other, as do the strains of serotypes reactions at the cell surface (532). The outer and c, e, and f. This observation corresponds to the inner structure of the cell wall of S. mutans is genetic subdivision (95), as discussed above. seen in Fig. 2. The protoplast membrane is not clearly defined. Reactivity of S. mutans with Lectins Fluorescein-labeled antibodies specific for pol- ysaccharide or a protein component give a uni- Plant lectins (phytohemagglutinins) have form surface stain of group A (89) and group F been found to react specifically with sugar resi- (619) streptococci. Ferritin-labeled antibody to dues of polysaccharides and glycoproteins (359). the group A streptococcal M protein (602) and The lectins bind to the surface components of group C polysaccharide (602a), and the LTA of microbial cells, frequently resulting in aggluti- S. mutans (588), show a surface location of these nation (217, 317, 318). antigens. The rapid agglutination of some S. It was shown that ConA, a jack bean lectin mutans strains by ConA indicates a similar lo- reactive with a-D-glucopyranosyl or a-D-man- cation of the polysaccharides (217). The binding nopyranosyl residues, agglutinated the cells of of bacteriophage by the peptidoglycan of the 13 of 15 strains of S. mutans in an 18-h incuba- group A streptococcus indicates a ready acces- tion (217). Among these, type a, d, f, and g sibility of the latter to the virus (82). strains agglutinated within 2 h. Cells of seven S. These data support the concept that these sanguis group H streptococcal strains and var- antigenic polymers are available at the cell sur- ious other bacterial species were also aggluti- face to react with other polymers. Electron mi- nated within 2 h by ConA. Binding of ConA to crographs ofstreptococci show filamentous-type the surface of serotype f S. mutans was con- structures on the cell surface (130, 432, 490, 558) firmed using 3H-labeled ConA (217). Further- which react with ferritin-labeled antibody (558, more, ConA will also induce the agglutination of 588, 602). Selective extraction procedures have S. sanguis and S. faecium (287, 452). distinguished the M protein from the teichoic Ricinus communis agglutinins (RCA I and acid structures at the cell surface of the group A RCA II), castor bean lectins reactive with galac- streptococcus (13). These filamentous structures tose residues (441, 442), agglutinated cells of may be termed "fimbriae." serotype a, d, and g S. mutans, but not those of A model of the structure of the streptococcal serotype b, c, e, and f S. mutans, after a 2-h cell wall is shown in Fig. 3 (532). The lattice-like incubation (217). cross-linking of the peptidoglycan is shown, al- These results with S. mutans indicate that the though all the possible linkages are not illus- two lectins react with a surface polysaccharide trated for purposes of clarity. The teichoic acid polymer. The binding of ConA, however, did not and LTA are shown in transit across the wall inhibit the binding of GTase to heat-treated S. (618) from the site of synthesis in the membrane mutans cells and subsequent adherence due to (524, 533). Protein and polysaccharide may also glucan synthesis (217). If binding of ConA did be synthesized in the membrane. Ferritin-la- occur at the type-specific polysaccharide or glu- beled antibody studies show that the fimbriae can sites, the lectin did not occupy a position are composed mainly of protein, polysaccharide, which affected the action of GTase. Many other and teichoic acid. Open spaces between the fun- lectins with different specificities (359) will pos- briae to the peptidoglycan may allow the binding sibly bind to and agglutinate the cells of various of bacteriophage. The close association of these oral streptococci. On the other hand, Staat et al. polymers in the wall helps to explain the release (546) found that Persea americana lectin in- of both polysaccharide and protein by proteo- VOL. 44, 1980 STREPTOCOCCUS MUTANS 341 V.W

.~~~~ ~ .f _I .I \ 'I l1I C, FIG. 2. Electron micrograph of thin section of S. mutans, serotype d, strain B13. F, fimbriae; OW, outer wall; IW, inner wall; C, cytoplasm. x125,000.

TA

PRX,X'S IMf' t'#;,.PS

Z--LTA"Al

PEPTIDOGLYCAN\

CYTOPLASMIC_.. MEMBRANE FIG. 3. Model of the streptococcal cell wall. PR, protein; LTA; lipoteichoic acid; TA, teichoic acid; PS, polysaccharide. (Reproduced with pernission, reference 532.) lytic and saccharolytic enzymes (179), the "um- designated "peptidoglycan" (513). The chemical brella" effect of antibody (531), and their func- composition of the cell walls of S. mutans has tion as binding sites for enzymes and sites of been reported (22, 272, 532). S. mutans pepti- enzymatic glucan synthesis in in vitro and in doglycan consistently contains glutamic acid, al- vivo adherence (13, 130, 531). aninq, lysine, glucosamine, and muramic acid in The rigid nature of the bacterial cell wall is the approximate molar ratio of 1:2-4:1:1:1. In due in large measure to a bag-shaped macro- addition to these major amino acids, the pres- molecule (606) which is composed of N-acetyl ence of threonine was reported in the cell walls amino sugars plus N-acetylmuramic acid and of serotypes a (22, 272, 534), d (22), and g (272). numerous peptides. This basal polymer has been The molar ratio of threonine to glutamic acid in 342 HAMADA AND SLADE MICROBIOL. REV. these cell walls is approximately 0.7-1:1. These charides, namely, glucans and fructans, from data indicate that most S. mutans strains con- sucrose by the enzymatic action of GTase (EC tain a peptidoglycan cross-linked by interpeptide 2.4.1.5) and fructosyltransferase (FTase; EC bridges consisting of L-alanyl oligopeptide or 2.4.1.10). These polysaccharides, especially glu- threonyl-alanyl peptide. Figure 4 shows a possi- cans, are considered to be critically important in ble structure of the peptidoglycan of S. mutans dental plaque formation and hence in the path- BHT (serotype b) proposed by Inoue et al. (271). ogenesis of dental caries, because they are water The bonds probably hydrolyzed by the Flavo- insoluble and possess a marked ability to pro- bacterium cell wall lytic enzymes are indicated. mote adherence when synthesized de novo on Figure 2 also illustrates the filamentous struc- various solid surfaces (Fig. 5). Since various as- tures on the surface of S. mutans. Their chemi- pects of S. mutans glucan have been reviewed cal nature has not been defined. Those of group recently (204, 233, 436, 603), only certain topics A streptococci are individually either protein or will be discussed briefly. teichoic acid (13). These structures on S. mu- Glucans. In general, all bacterial glucans con- tans, seen on practically all gram-positive cocci, tain a(l -- 6) and a(1 -- 3) glucosidic linkages, have been termed "fuzzy coat" (432). We prefer with the occasional occurrence of a(1 -- 2) or the term "fimbriae," although they are not iden- a(l -- 4) linkages. It has been reported that the tical to the structures on the gram-negative ba- proportion of a(1 -- 3) linkages varies from 0.5 cilli. The latter are frequently termed "pili" to 60%, depending on the origin of the glucan (281a). (603). ConA binds native glucans to form a pre- Proteins other than those associated with the cipitate, and branched glucans have a higher serotype b antigen also exist in the cell wall of S. affinity for ConA than do the linear ones (dex- mutans. A protein which functions as a binding trans) (483). site for glucan has been isolated (387, 391). Evi- Earlier investigations (182,212) indicated that dence of proteins which act as binding sites for extracellular polysaccharides produced by S. GTase or glucan has been presented (178, 333, mutans were an a(1 -- 6)-linked linear "dex- 427, 428, 622, 623). Further studies will probably tran." In fact, water-soluble glucan from S. mu- identify other active proteins of this type. tans has been reported to consist of an a(1 -* 6)-linked linear glucose polymer with a(1 -- 3) POLYMER SYNTHESIS BY S. MUTANS glucosidic branch linkages (376). However, most of the S. mutans glucan in Extracellular Polysaccharides sucrose-containing broth is in a cell-associated S. mutans synthesizes extracellular polysac- form, which is essentially water soluble. The

/ MU/ / 1 (a) MUrNAC /1-Ala--. D-G u-NH2 / <~ (a) / 1Y) / -Al a-D-Gl u-NH2 GicNAc L-Lys_-.D-Ala-COOH 40() 1, 4 GlcNAc L-Lys-._D-Ala-- L-Ala--_ (L-Al a) -2 .-D-Al a -- L-Al a _+ ( L-Al a) -0 2

MurNAc / / s~~~'1 (a) MurNAc /L-Ala -_ D-Gl u-NH2 / <:t (a) / (Y) / -Ala -_D-Glu-NH2 GlcNAc L-Lys-COOH

G1cNAc L-Lys _-D-Ala - L-Al a-_ (L -Al a)O - 2 1 Et / ----'D-Ala -- L-Ala--.(L-Ala)02 t /1 / FIG. 4. Possible structures ofpeptidoglycan ofS. mutans strains BHT ceU walls and points of attack of a cell wall lytic enzyme from Flavobacterium sp. strain L-11. GlcNAc, N-acetylglucosamine; MurNAc, N- acetylmuramic acid; Glu, glutamic acid; Ala, alanine; Lys, lysine. Horizontal arrows, N-acetylmuramyl-L- alanine amidase; vertical arrows, D-alanyl-L-alanine endopeptidase. (Reproduced withpermission, reference 271) FIG. 5. Scanning electron micrograph of S. mutans strain OMZ176 (serotype d) grown in glucose broth (top) and sucrose broth (bottom). Cells grown in thepresence ofsucrose were covered with amorphous capsule- like material of heavy thickness which was adherent to a glass surface. (Reproduced with permission, reference 224) 343 344 HAMADA AND SLADE MICROBIOL. REV. insoluble glucan can be extracted in an alkaline relation to the water-insoluble ones remain to be solution, followed by ethanol precipitation. The elucidated. precipitate can be separated into structurally Fructans. Certain strains of S. mutans have different water-soluble and insoluble fractions. been reported to synthesize fructans in addition The latter possesses more a(1-3 3) glucosidic to glucans from sucrose (56, 124, 224, 427, 474, linkages than does the former (163, 443, 581) and 507). In the earlier phase of these studies, the is more resistant to the enzymatic action of a(1 fructan was considered to be levan consisting of 6) glucanase, i.e., dextranase (224). a(2 -- 6) fructofuranoside linkages. Insoluble glucan can be more conveniently Baird et al. (8) suggested that the predomi- obtained by incubating cell-free GTase and su- nant linkage of the fructan from S. mutans was crose. The glucan is obtained by centrifugation an inulin-type ,8(2 -- 1) fructofuranoside linkage and then washed extensively with water and rather than fl(2 -- 6). This has been confirmed lyophilized. Using the glucan thus obtained, by other investigators (19, 124, 491). S. mutans Guggenheim (203) showed that water-insoluble fructans occur in both water-soluble and water- glucan from S. mutans strain OMZ176 contains insoluble states (124, 507), and the production of a markedly high proportion (up to about 90%) fructans appears to differ from strain to strain, of a(1-- 3) glucosidic linkages. He proposed the depending on cultural conditions. name "mutan" to distinguish this water-insolu- ble glucan from typical linear-linked "dextran." Polysaccharide-Synthesizing Enzymes In contrast to the insoluble glucan from S. mu- S. mutans can produce extracellular GTase or tans, gelatinous glucan from S. sanguis strain FTase constitutively (609), which allows the syn- 804 has equal amounts of a(1 -- 3) and a(1 -- 6) thesis of water-soluble, adherent glucans in ad- glucosidic linkages (203). dition to certain amounts of fructans from su- Ample evidence has accumulated supporting crose (233). Notwithstanding the complex chem- the original finding by Guggenheim (203) regard- ical structure of glucans, only GTase(s) appears ing the chemical structure of water-insoluble to be responsible for the synthesis of glucan. It glucans from various strains of S. mutans as well catalyzes the transfer of a glucosyl moiety from as other oral streptococcal species (8, 65, 125, sucrose to a terminal site on the growing glucan 247, 384, 444). It appears that the large propor- molecule: tion of a(1-+ 3) glucosidic linkages found in the . -- insoluble glucan explains the insoluble nature of n sucrose (glucose),, + n this polymer. The techniques of periodate oxi- The equilibrium of this reaction is almost irre- dation, Smith degradation, and methylation versibly to the right. Practically, sucrose is the analysis have shown that the consecutive a(1 sole substrate for GTase. However, Figure and 3) glucosidic linkages form long chains as the Edwards (148) have shown that a-D-glucosyl backbone of a highly branched insoluble glucan can act as the donor for GTase of S. (125, 247). A similar type of a(1-- 3) glucan mutans FAl to synthesize insoluble glucans. from the cell walls of certain fungi is a structural Purification of S. mutans GTase has been component and is also water insoluble (6). attempted in various laboratories. Guggenheim There are some significant differences in the and Newbrun (209) obtained three major GTase quantities and chemical nature of the extracel- fractions from the supernatant fluid of a culture lular glucans synthesized by various serotypes. of strain OMZ176 (serotype d), using hydroxy- According to Trautner et al. (582), their "type apatite (HA) chromatography followed by iso- d" S. mutans strains synthesized significantly electric focusing. These fractions possessed dif- higher amounts of glucans than the "type c" ferent isoelectric points (pH 4.24 to 5.65) and strains, and the ratio of insoluble to soluble different pH optima (pH 5 to 7). The multiple glucans was higher with the "type d" strains. nature of GTase may account for the heteroge- The difference is explained by the presence of neity of the product glucans (75). the higher proportion of a(1-- 3) linkages in the On the other hand, Fukui et al. (170) separated "type d" glucans (580), as is discussed above. GTase and invertase, another sucrose-splitting Glucans produced by incubating sucrose and enzyme, from culture supernatant of S. mutans cell-free GTase of S. mutans strains (serotypes HS6 (serotype a). GTase was separated into two a to g) can be separated further into one water- fractions by agarose chromatography. The insoluble and three soluble fractions. Each frac- lower-molecular-weight fraction synthesized wa- tion possesses a different content of a(1-. 3) ter-soluble glucan, whereas the higher-molecu- glucosidic linkage, a different molecular weight, lar-weight fraction synthesized water-insoluble and a different reactivity with ConA or S. mu- glucans. Mukasa and Slade (429) obtained a tans cells (273). The regulatory mechanisms for GTase fraction synthesizing insoluble adherent the synthesis of water-soluble glucans and their glucan from the same strain and another fraction VOL. 44, 1980 STREPTOCOCCUS MUTANS 345 synthesizing water-soluble glucan. The former 2.4.la), have been shown to be involved in IPS preparation contained significant portions ofglu- synthesis in S. mutans (20, 240). ADP-glucose is cose polymer, suggesting that the enzyme resem- synthesized from adenosine triphosphate and bles a glycoprotein. Similar findings have been glucose 1-phosphate by the former enzyme, and obtained with the serotype c strain GS5 GTase the latter enzyme catalyzes formation of glyco- (330) and the serotype b strain FAl GTase (500). gen, using ADP-glucose as the glucosyl donor. More recently, Mohan et al. (421) have IPS metabolism appears to be influenced suggested that soluble and insoluble glucan mainly by the pH of the external environment syntheses are catalyzed by interconvertible (164). S. mutans will produce ethanol and acetic forms of the same enzyme protein. acid in addition to from IPS under A highly active GTase fraction obtained from the limitation of exogenous glucose, whereas strain 6715 (serotype g) contained two separate only lactic acid is formed in the presence of bands on polyacrylamide gel electrophoresis excess glucose (262). (71). The purified GTase fraction had 30 to 40% In the deep region of plaque, the cell walls of carbohydrate, which coincides with the finding gram-positive coccal bacteria become thickened described above. The GTase activity was also and the majority of the cells contain scattered found to be completely dependent upon a primer IPS granules in the cytoplasm. On the other dextran (176). Also, purified GTase was resolved hand, cells located in the superficial portion of into two different components which were re- the plaque possess normal cell wall morphology sponsible for the synthesis of water-soluble and and fewer IPS granules (597). Rifampin treat- water-insoluble glucans, respectively (73, 429). ment of S. mutans cultures results in accumu- High concentrations of mono- and divalent lation of IPS and thickening of the cell walls cations promote the synthesis of insoluble glu- accompanying inhibition of ribonucleic acid can by an enzyme from strain 6715 (424). De- (RNA) synthesis (383), whereas tetracycline tailed biochemical properties of the GTase en- treatment causes cell wall thickening accompa- zyme from various S. mutans strains have been nying inhibition of protein synthesis but little reviewed elsewhere (233, 436). accumulation of IPS (384). These findings indi- cate that IPS synthesis may be influenced by Intracellular Polysaccharides various cultural conditions. Many plaque bacteria can synthesize intracel- lular iodine-staining polysaccharides (IPS) from Lipoteichoic Acid high concentrations of various sugars. Most S. LTAs are a glycerol form of teichoic acid mutans strains produce a storage IPS which covalently linked to a lipid moiety (616, 617). may contribute to the pathogenicity of S. mu- LTA occurs as a cellular surface component and tans (16, 17, 191, 594). Stored IPS may be the extracellular product of a number of gram-posi- source of acid when exogenous sugar is not suf- tive bacterial species, including all serotypes of ficient or is absent. S. mutans (90, 223, 285, 381, 519). PGP is the Among various serotypes of S. mutans, sero- backbone structure of LTA and is responsible type d and g strains produce and metabolize less for a common antigenic specificity among S. IPS than serotype c and e strains. IPS-synthe- mutans (72, 234, 311, 312, 395). The amphipathic sizing strains degrade the IPS to produce acid(s) nature of LTA strongly influences the immuno- when external carbohydrates are deprived (164). biological activities of this unique polymer. LTA It is reported that strains 6715 (serotype g) and possesses most of the biological activities of the OMZ176 (serotype cl), like other serotype d and lipopolysaccharides of gram-negative bacteria. g strains, produce little or less IPS, whereas they Among these are immunogenicity, spontaneous produce marked dental caries in experimental sensitization of erythrocytes, bone resorbing ac- animals (111, 202, 229, 567). Therefore, IPS ap- tivity in organ culture, complement fixation, and pears not to be a prerequisite for the cariogen- stimulation of nonspecific immunity (249, 618). icity of S. mutans. Mutants of serotype c strains LTAs and certain lipids have also been found to which are weak in their ability to synthesize IPS inhibit cellular autolysis of S. faecalis (83). The show diminished cariogenic activity (166, 568). general features and biological characteristics of IPS is a glycogen-like glucan with a(l -* 4) LTAs have been extensively reviewed (21, 321, and a(1 -* 6) linkages which are susceptible to 313, 617, 618). a-amylase (99, 596). IPS forms a complex with Evidence indicates that LTAs are closely as- I2-KI and produces a brownish-yellow color with sociated with the cytoplasmic membrane (524, an adsorption maximum at 520 nm (111). 533), and they were frequently called "mem- Two enzymes, adenosine diphosphate (ADP)- brane teichoic acids" or "intracellular teichoic glucose pyrophosphorylase (EC 2.7.7b) and acids." It appears that membrane association ADP-glucose-glycogen glucosyltransferase (EC depends upon a covalent linkage between PGP 346 HAMADA AND SLADE MICROBIOL. REV. and a glycolipid protein of the membrane. An branes (phospholipid vesicles). The latter are electron microscope study using the ferritin-la- very effective in separating various contami- beled antibody technique revealed membrane nants from cellular and extracellular acylated association more directly in Lactobacillus fer- LTAs of S. mutans strains. menti and L. casei (588). It was further demon- There is much speculation concerning the pos- strated that the label extended from the outer sible functions of LTAs in mammalian tissue membrane layer through the matrix of the cell and the organism itself (21, 83, 259, 618). We wall beyond the cell surface and into the external have found that almost all of the hemagglutin- environment (617, 618). Chorpenning et al. (72) ating antigen in the culture supernatant of S. reported that glycerol teichoic acid was found in mutans strains can be recovered by 50% ammo- a phenol extract from purified cell walls of S. nium sulfate precipitation. Immunological tests mutans. Furthermore, a muramidase (mutano- using an antibody specific for PGP demonstrate lysin) lysate of S. mutans cell walls reacted with that the hemagglutinating activity is due to ex- antiserum specific for PGP (S. Hamada, unpub- tracellular LTAs. The extracellular LTA is lished data), indicating the "presence" of glyc- closely associated with glucosyltransferase activ- erol teichoic acid or even LTA in the cell walls. ity, and it is difficult to separate the two (223). Extracellular LTAs are found in the cell-free Both cellular and extracellular LTAs are effec- culture supernatant of many gram-positive bac- tively adsorbed to HA powder (76, 223). Phos- teria, and in particularly high amounts in S. phate and fluoride inhibit adsorption of LTA. mutans strains (285, 381). The greatest recovery Phosphorus contamination of extracellular glu- of extracellular LTA was obtained by S. mutans cans synthesized by oral streptococci (404) may organisms growing at a low dilution rate at pH be explained by the strong affinity between glu- 6 to 6.5 in a chemostat (276). It is likely that cosyltransferase and extracellular LTA mole- extracellular LTAs are present as a result of cules, thus forming complexes of glucan-LTA- active excretion rather than cellular lysis or as glucosyltransferase. It has also been shown that a result of turnover during cell growth. It thus sucrose-grown S. mutans binds higher amounts appears that LTAs are in transit from a cellular of calcium than do glucose-grown cells. In light to an extracellular location in the S. mutans cell of these findings, it is proposed (484-486) that (285, 301). the calcium-binding ability of LTA should be a Both cellular and extracellular LTAs can usu- major selective factor in the adherence of gram- ally be separated into two peaks by Sepharose positive bacteria to enamel surfaces. It should 6B gel filtration. One is the acylated form, and be added here that oral streptococcal strains do the other is in the deacylated form. Only the not necessarily have LTA. It has been reported former LTA can sensitize erythrocytes, and it that a considerable number of S. mitior (489) contains higher levels of fatty acids (381). Both and biotype B S. sanguis (S. Hamada and J. components are synthesized and excreted by Mizuno, unpublished data) strains lack LTA. logarithmically growing cells of S. mutans. Furthermore, a new amphipathic antigen has However, in contrast to the LTA from S. been isolated from Actinomyces viscosus (615). mutans, only deacylated LTA is detected in the LTA-negative S. sanguis also has an erythro- culture fluid of an S. faecium strain. Most prob- cyte-sensitizing antigen which is immunologi- ably this extracellular deacylated LTA is derived cally different from LTA/PGP (Hamada and from cellular LTA by enzymatic deacylation Mizuno, unpublished data). (300). Most investigators conveniently use a phenol- Interaction of Glucosyltransferase water extraction method to obtain "native" with Various Agents LTAs from bacterial cells (21, 90, 223, 312, 313, The distribution of GTase in broth cultures of 423, 616, 617). Various extraction methods also S. mutans is strongly influenced by various fac- yield an antigenic component in the extract tors. In many cases, almost all of the GTase which reacts with antibody specific for the PGP activities are found extracellularly in sucrose- backbone of LTA. However, drastic procedures free media (237, 389, 474), although the occur- usually split the linkage between PGP and the rence of significant cell-associated GTase activ- lipid moiety or cause the hydrolysis of phospho- ity in addition to cell-free GTase activity is diester linkages (223, 312). LTA is also released reported by some investigators (187, 298, 277, from S. mutans cells by treatment with leuko- 278, 329, 422). Ample evidence indicates that the cyte hydrolases or with lysozyme or phenol presence of, or the addition to culture media of, (518). sucrose results in the synthesis of cell-associated More recently, Silvestri et al. (526) developed GTase (232, 237, 389, 474, 544). a more refined method to purify LTAs by using McCabe and Smith (389) consider that GTase gel flltration, hydrophobic interaction chroma- is reversibly bound to the insoluble glucan dur- tography, and adsorption to synthetic mem- ing the synthesis of the glucan by sucrose-grown VOL. 44, 1980 STREPTOCOCCUS MUTANS 347 S. mutans cells. The enzyme then becomes ir- fold increase in water-soluble glucan synthesis reversibly bound, and is finally inactivated as by S. mutans GTase. The increased rates of insoluble glucan accumulates. Reversibly bound glucan synthesis by lysophosphatidylcholine GTase can be eluted in solutions of clinical dex- and primer dextran are additive (248). In a sub- tran or guanidine hydrochloride. This property sequent study, phospholipids normally detected is used to carry out affinity chromatography for in human oral fluids, e.g., saliva from various purification of GTase (274, 391, 392, 541). glands, gingival crevicular fluids, and serum, en- Some commerical media, such as Todd-Hew- hanced the activity of GTase (503). The GTase itt broth and Trypticase soy broth (BBL), con- level is reported to be increased about fivefold tain trace amounts of sucrose; when S. mutans in the presence of 1.0% Tween 80; alteration of was grown in these media, essentially all GTase the fatty acid composition of the S. mutans cells activity was in a cell-associated form (237). Pre- also occurs (583). Many other nonionic surfac- treatment of these culture media with yeast tants promote the activity of GTase, whereas invertase before autoclaving resulted in an S. anionic and cationic surfactants inhibit this ac- mutans culture which contained increased tivity. Lower concentrations of ampholytic sur- amounts of extracellular GTase. Related to this, factants activate GTase activity; this is followed most of the GTase activity produced by S. mu- by almost complete inhibition of GTase at high tans grown in sucrose-free chemically defined concentrations (0.1% or more) of the surfactants medium has been found to be extracellular; this (M. Torii and S. Hamada, unpublished data). was in contrast with growth on Todd-Hewitt Enhanced levels of cell-free GTase are also ob- broth or Trypticase soy broth (232, 277, 505). In tained when cells of S. mutans are grown in view of these findings, extracellular and cell- penicillin (100 to 250 ,ug/ml)-containing medium associated GTases are most likely alternate (278). This may be due to release of certain lipid states of the same enzyme protein. components from the cells by an unknown mech- The addition ofsoluble dextran stimulates the anism (258) which in turn results in an enhance- reaction of GTase with sucrose (170, 176, 330, ment of GTase levels. 389,429). This activation is due to a requirement by GTase for a primer molecule; its nonreducing Invertase ends are required for new glucan synthesis. The Invertase (,8-fructofuranosidase; EC 3.2.1.26) primer may also act as the site to which newly is a sucrase that catalyzes the hydrolysis of the synthesized units are added (178). Equal weights glucosidic linkage of sucrose, which results in of with different molecular weights the release of an equimolar ratio of glucose and show a similar priming effect on new glucan fructose. synthesis by GTase (176, 238). On the other Gibbons (180) first suggested the presence of hand, Robyt and Corrigan (475) have reported an intracellular, inducible "sucrase" activity that the activation of GTase by dextran cannot other than GTase and FTase in S. mutans GS5 be due to a primer reaction with the nonreducing (serotype c). Toluene treatment of intact cells end because of the nonavailability of the non- that destroys the selective permeability system reducing ends of the dextrans chemically modi- of the bacterial cell membrane (338) revealed fied by reaction with trypsyl chloride or hydrol- enhanced invertase levels in S. mutans GS5 and ysis with an exodextranase (475). KlR (serotype g) (180, 393). The molecular The addition of increasing amounts of soluble weight of intracellular invertase has been cal- dextran will cause a decrease in the synthesis of culated to be 47,000 to 48,000, and the invertase insoluble glucan and an increase in the synthesis has a relatively high Km value for sucrose (35 to of soluble glucan (422, 475). Certain sugars such 140 mM) (327, 564). The intracellular location of as maltose and fructose significantly reduce the the invertase implies the presence of a sucrose yield of insoluble glucan (238). permease system, but little is known about the The enhancement of GTase activity by var- sucrose transport mechanism ofS. mutans. Con- ious humoral fluids such as rabbit antiserum troversial results have been obtained on the (139), rat oral fluid (45), and monkey antiserum inducibility of invertase from various strains of (26) has been reported. S. mutans (327, 393, 564). The biochemical prop- Fukui et al. (169) reported that the secretory erties of the invertases found in the 37,000 x g component ofsecretory immunoglobulin A (IgA) soluble cell fraction are different in the individ- caused a severalfold acceleration ofGTase activ- ual serotypes of S. mutans but similar within ity as compared with the control without addi- the same serotypes (565). Invertases from sero- tives. However, results with purified secretory types e, f, and g are reported to be structurally IgA component have not confirmed this study similar to that from serotype c (385). (88). Extracellular invertase also exists (70, 170) More recently, lysophosphatidylcholine, a and has a molecular weight of 5 x 105 (385). phosphoglyceride, has been found to cause a 2.6- The physiological role of S. mutans invertase 348 HAMADA AND SLADE MICROBIOL. REV. is not fully understood. However, a large portion of formate, acetate, and ethanol in addition to ofavailable sucrose is hydrolyzed by this enzyme lactate when glucose is limiting (61). An in vivo (69). The remainder of the sucrose is converted study supports the latter finding (584). to the synthesis of glucan or fructan via GTase Sucrose has also been shown to serve as the or FTase. energy source during growth of S. mutans in Recently, strains of S. mutans belonging to addition to its role as the substrate for extracel- various serotypes have been found to have lular glucan synthesis. Most of the glucosyls of highly efficient phosphoenolpyruvate (PEP)-de- sucrose are converted into lactic acid. Only a pendent sucrose phosphotransferase activity small portion of sucrose is diverted to extracel- that can initiate the catabolism of sucrose and lular polysaccharide synthesis (473, 562, 566). produce sucrose phosphate. The Km value of the Furthermore, S. mutans is known to utilize su- enzyme for sucrose is reported to be about 70 crose at a significantly faster rate than other oral 1iM, indicating that the enzyme, unlike invertase, bacteria such as S. sanguis, S. mitis, and Acti- can function even at low substrate conditions nomyces viscosus (418,450). S. mutans produces (499, 535). significant amounts of intracellular polysaccha- ride from sucrose, which can be converted to a(1-- 6) Glucanase lactic acid after prolonged incubation (419). The a(1 -3 6) Glucanase [a(l -. 6)-glucosidase; EC organism also produces mannitol when high lev- 3.2.1.11] is synthesized constitutively by some els of sucrose or glucose are present (370). Com- strains of S. mutans as well as certain other parison of metabolic activities of "cariogenic" bacterial species in dental plaque (110, 509, 548). and "noncariogenic" plaques indicates that S. An endoglucanase specific for the a(1 -* 6) mutans is metabolically dominant in plaques linkage has been purified from the culture su- closely associated with the carious lesion (418). pernatant of S. mutans OMZ176 (serotype d) S. mutans is more aciduric than other oral strep- (205). The biochemical property of the enzyme tococcal species (114). is similar to that of mold dextranases in general. In the presence of sucrose, S. mutans grows at A similar endo-a(1-- 6) glucanase was obtained the same exponential rate as it does on glucose from the culture supernatant of serotype g (100). A previous finding that growth of S. mu- strains 6715 (131, 177) and KlR (465, 466). tans is linear in sucrose culture (572) is attrib- These results indicate that most glucans pro- uted to an optical artifact based on the formation duced by growing S. mutans cells or by "crude" of visible cell aggregates (100). GTase preparations obtained from culture fluids S. mutans transports glucose into cells via a could be synthesized under the influence of in- membrane-associated PEP-dependent phospho- trinsic or contaminating a(1 -- 6) glucanase. transferase system (132, 241, 501, 508). Sucrose Therefore, structural heterogeneity ofthe S. mu- and lactose are similarly transported in S. mu- tans glucans may be a result of the combined tans by this system (49, 499, 535). The nonfer- enzymatic action of GTase and endoglucanase mentable glucose analog D-2-deoxyglucose is an activities. Relative quantities of these enzymes effective inhibitor of glucose transport by the S. may significantly affect the chemical and phys- mutans PEP-dependent glucose phosphotrans- ical properties of glucans synthesized by S. mu- ferase system (506). tans. In addition to the polymers described above, ADHERENCE OF S. MUTANS S. mutans produces proteases active against ca- The adherence of S. mutans and other oral sein and glycoprotein (93) and against collagen bacteria to tooth surfaces and the formation of (492), phospholipase A (44), and arylaminopep- dental plaque are of major significance in the tidase (456). S. mutans also produces intracel- development of dental caries. These processes lular hydroxyapatite crystals which may be re- are complex and involve a variety of bacterial sponsible for calculus formation (552). and host components. Various aspects of bacte- rial adherence in the oral cavity have been ex- SUGAR METABOLISM BY S. MUTANS tensively reviewed (194, 195, 233, 530, 531, 589). S. mutans has been reported to be a homofer- mentative lactic acid bacterium (115, 282, 569). Initial Attachment of S. mutans to However, the metabolic pathway of glucose by Smooth Surfaces S. mutans varies, depending on environmental Bacterial attachment to the tooth surface is factors. The major fermentation product of S. usually preceded by the formation ofan acquired mutans is lactate, especially when the organism pellicle of salivary origin. The initial stages of is grown in the presence of excess glucose, plaque development on cleaned tooth surfaces whereas S. mutans produces significant amounts require cell attachment to the pellicle suffi- VOL. 44, 1980 STREPTOCOCCUS MUTANS 349 ciently firm to resist local cleansing forces of Interaction of Salivary Components salivary flow and muscular movements. The at- with Streptococcal Cells tachment may involve specific interaction ofpel- licle components with selected bacterial species. Direct interaction ofsalivary components with 0rstavik et al. (454) found a significant in- bacterial cells seems to be significant in regulat- crease in attachment of S. mutans, S. sanguis, ing the attachment and accumulation of differ- and S. salivarius to pellicle-coated enamel slabs ent bacterial species involved in plaque forma- when compared with an uncoated slab. The in tion. Whole saliva is known to possess the ability vitro adherence of S. sanguis was significantly to agglutinate many plaque bacteria (193). The greater than that of S. salivarius, and both salivary agglutinating factor was reported to be species adhered in greater numbers than did S. a high-molecular-weight glycoprotein which is mutans. heat-stable and Ca2" dependent and which oc- S. mutans has been found to attach in greater curs optimally between pH 5 to 7.5 (193, 250). It numbers to dextran-coated HA than to pellicle- is unlikely that IgA is involved in these interac- coated or uncoated HA, whereas the attachment tions (136, 288, 620). Different agglutinating fac- ofS. sanguis and S. mitior was not enhanced by tors have been found for S. mutans, S. mitior, dextran-coated HA (350, 351). The in vitro affin- and S. sanguis (136, 194, 286). When saliva is ity of each oral streptococcal species for pellicle- pretreated with wheat germ agglutinin, the sa- coated solid surfaces appears to correlate with liva does not induce the agglutination of S. mu- the proportions of that species found in vivo tans strains. N-Acetylglucosamine, forwhich the (195). It is likely that S. mutans does not play a lectin shows a specificity, does not block the key role in the initial stages oftooth colonization, inhibitory effect of the lectin (420). although the latter experiment was performed These salivary agglutinating factors may re- in the absence of sucrose. semble lectins in that specific determinants may Recently, a model has been proposed by Rolla bind selected bacterial species. The salivary ag- (486) suggesting that cells of S. mutans and S. glutinating factor that is responsible for binding sanguis behave like negatively charged particles S. sanguis was destroyed by neuraminidase or in their electrostatic interaction with HA sur- protease treatment, indicating the importance of faces in vitro. He demonstrated that calcium sialic acids (386). The mucin-like glycoprotein and protamine phosphate significantly increased agglutinated both S. mutans (serotypes b and d) uptake ofbacteria, whereas , phosphate, and S. sanguis strains ATCC 10556 and 10558 or even saliva decreased the uptake of the cells. (347). Interestingly, elimination of sialic acid The acidic proteins in saliva are selectively from the glycoproteins resulted in a loss of ag- bound by HA. It is considered that pellicle for- glutination of S. sanguis but not of S. mutans. mation by acidic protein results in a reduction It is suggested that salivary lysosyme may par- of the cationic nature of the surface and reduces ticipate in the agglutination of some S. mutans the binding of bacterial cells. strains (463). It appears that a large number of hydroxyl Preincubation of various bacteria and saliva groups on the surface of sucrose-grown S. mu- reduced the attachment of bacteria to HA sur- tans and S. sanguis cells preferentially form faces (78, 80, 379). Serotype c S. mutans cells hydrogen bonds with the pellicle proteins (487). appeared to bind salivary components (78, 379). LTAs are closely associated with extracellular Therefore, it is suggested that the agglutinating GTase (223) and its product glucan (76, 404). factor free in saliva competitively inhibits the LTAs possess a strong affinity for HA (486). interaction between salivary coated HA and Divalent cations such as Ca2" were found to those surface components of the bacterial cells enhance the interaction between a negatively which contain bound salivary glycoproteins. charged pellicle surface and a similarly charged Recently, Gibbons and Qureshi (189, 190) bacterial cell surface (293, 484). Related to this, found that strains of S. mutans and other oral ethylenediaminetetraacetic acid, EDTA, is bacteria bind the blood group-reactive (BGR) known to have a strong plaque-disintegrating mucins of saliva after exposure to whole saliva ability (293, 369); this supports the concept that or partially purified mucin preparations. Differ- calcium bridges are essential for the initial bind- ent serotypes of S. mutans bind different com- ing of the bacterial cell to the pellicle surface. ponents of BGR mucins. BGR salivary mucins The major discrepancy between the reports of are present in the acquired pellicle on the tooth Rolla (486) and those of Liljemark and Schauer surface (542), which may serve as receptor mol- (350, 352) and 0rstavik et al. (454) may be ecules involved in the attachment of bacteria to ascribed to the use of buffers of high ionic teeth, suggesting that a lectin-receptor-type strength in the case of the latter investigations. mechanism is involved (181). 350 HAMADA AND SLADE MICROBIOL. REV. Implantation of S. mutans More recently (600), the colonization of S. Active transmission of S. mutans im- mutans 6715 occurred in rats fed diets with a implies sucrose content from 56% to as low as 1%, in plantation in a receptive host. However, S. mu- tans is not easily implanted in adult humans. which the lowest effective inoculum was 105 in the of S. mutans to CFU by a single oral administration (600). More Variability ability implant frequent inoculations with about 5 x 108 CFU in different has been a subjects noted, and grad- were needed to establish the organisms on a ual decrease in the number of orga- implanted high-glucose diet. When inoculated with less nisms has been observed over an extended pe- than 107 CFU, however, the cells were gradually riod of time (284, 323). eliminated from the teeth. When S. mutans was onto implanted human It should be noted here that streptomycin- tooth surfaces, the bacterium was recovered in much higher numbers from originally pellicle- resistant mutants of S. mutans frequently colo- nize less effectively than parent strains (12, 158). free than pellicle/plaque-coated tooth surfaces For (493). example, the minimum dose required for An in vivo with implantation ofa streptomycin-resistant mutant study human subjects has of S. mutans LM7 was more than 2 x 107 CFU, revealed that S. mutans can be recovered from whereas the minimum dose of the parent strain cleaned tooth surfaces after a few hours of oral was about 104 CFU (79, 591). The basis of the exposure when the salivary concentration of this phenomenon has not been well explained. bacterium exceeds a certain level. In adult hu- Preformed dextran/glucan, whether associ- man subjects with a salivary S. mutans count of ated with S. mutans cells or with the tooth about 104 colony-forming units (CFU)/ml or surface, does not permit the degree of cell at- less, the organisms could not be isolated from tachment that occurs in the presence of sucrose the tooth surface (593). On the other hand, at a (598). Apparently, de novo glucan synthesis (see salivary concentration of 103 CFU/ml, artificial next section) leads to a far stronger adherence fissures inserted in three adult subjects were to the tooth surface than that which occurs in colonized (555). Fluoride administration is un- the presence of dextran/glucan precoated on related to the colonization of S. mutans on hu- either tooth surface or bacterial cell surfaces, man teeth (590). In a later study (118), the although sucrose may not be indispensable to frequency of detection and concentration of S. the initial attachment of S. mutans in the oral mutans in saliva were higher in older children. cavity. In this connection, high numbers of S. However, some infants can acquire S. mutans mutans may be detected in the mouths of chil- shortly after tooth eruption (15, 382). dren with sucrase-isomaltase deficiency who, When organisms of serotypes a and c S. mu- therefore, consume a diet with an extremely low tans were implanted in humans, serotype a sucrose content (592). (strain E49) failed to colonize, although a sero- In rats, colonization of S. mutans occurred type c strain of human origin appeared to colo- with increasingly greater difficulty as the rats nize (556). The intraoral spread of the implanted became older (599). The actual mechanism re- S. mutans was confirmed on the adjacent and sponsible for the changes during aging remains antagonistic tooth surfaces (557). Furthermore, to be elucidated. However, the age effect was surfaces which harbored significant numbers of not observed when rats were fed a sucrose diet, S. mutans tended to remain positive, whereas whereas the results from rats fed a glucose diet surfaces which did not possess detectable num- indicate that changes may have occurred early bers of S. mutans remained at that level, indi- after weaning. cating that S. mutans does not evenly colonize In Macaca irus monkeys fed by stomach tube the surfaces of teeth (184). and provided with oral supplements, the colo- Sucrose-Dependent In Vivo Adherence nization of S. mutans was dependent upon su- of S. mutans crose from the drinking water. Withdrawal of Sucrose has been reported to markedly facili- the sucrose resulted in complete absence of de- tate the colonization of S. mutans on teeth. In tectable S. mutans on the teeth, although the early studies using hamsters and rats, it was salivary counts of S. mutans remained un- found that S. mutans could be established far changed (306). more easily when the animals were given su- crose-containing diets (128, 207, 319, 320). It was Sucrose-Dependent In Vitro Adherence also found that S. mutans could implant in the of S. mutans human oral cavity after inoculation with a pure Active glucan synthesis from sucrose has been culture, and the frequent chewing of sucrose found to foster the adherence of S. mutans to gum enhanced the implantation (128). various solid surfaces (195, 215, 233, 388, 427, VOL. 44, 1980 STREPTOCOCCUS MUTANS 351 428, 530, 531, 578). Synthesis of the glucan is mediated by the enzymatic action of cell-free XX (extracellular) or cell-bound GTases. This con- glucan-j ,XX cept is supported by the finding that mutants of iX "X- AX3 S. mutans which lack the ability to synthesize ,X' dextran-A,? water-insoluble, adherent glucans do not adhere to solid surfaces (107, 165, 314). Heat-treated cells of S. mutans which do not have cell-bound GTase adhered to a glass sur- face when incubated simultaneously with su- crose and exogenous GTase. No adherence oc- curred in the absence of de novo glucan synthsis / ~ ~ uA~~ "its ' (215, 427, 428). Later studies have clearly dem- onstrated that the adherence ofS. mutans in the presence of sucrose depends primarily upon the FIG. 6. Polysaccharide (PS) and protein (PR) as specific binding of extracellular GTase synthe- binding sites for GTase and the binding of dextran sized by S. mutans (218, 235, 237, 428, 530). The to protein on the surface of S. mutans. TA, lipotei- nonadherent property of other bacterial species choic acid. Symbols: 0, dextran-like site which in- volves binding of GTase; U, site which binds the is due to their inability to bind GTase to the cell dextran responsible for cell agglutination. (Repro- surface. However, a nonspecific adherence of duced with permission, reference 532.) cells of a variety of bacterial species can be obtained when the cells, GTase, and sucrose are incubated together (218, 530). This process, due to cell-free synthesis of glucan, is a nonspecific 15 trapping mechanism for adherence that may contribute to the development of dental plaque. 10. Glucan on the surface of S. mutans appears to function as a binding site for GTase (232, 237, ~~p-~-g;P- -4------4-0 333, 531). Antiserum against a glucan synthe- sized by a type c strain blocked binding of GTase and subsequent adherence (237, 427, 428). The strong affinity of GTase to glucan has been reported; however, adherence was not measured in these experiments (314, 389, 391, 392, 472). 0 0 40 60 80 00 Antiserum against the type a polysaccharide GTase (.uj) antigen also inhibited adherence. These results FIG. 7. Glucan synthesis from [14C]glucose-la- indicate that glucan may not be a specific bind- beled sucrose (59,000 dpm) by extracellular GTase ing site and that other complex polysaccharides bound to the surface of heat-treated S. mutans B13 may mediate the process. Protein is also in- (serotype d) cells and cell-free, water-insoluble glu- volved in the of GTase The cans and subsequent adherence to a glass surface. binding (428). gly- To heat-treated cells or insoluble glucans (1.0 mg, coprotein-like characteristics of GTase (531) dry weight) was added to 0 to 100 jil of extracellular may be related to its ability to bind to both GTase (specific activity, 56.6 mU/lI), and the mixture polysaccharide and protein molecules. Figure 6 was incubated for 10 min at 200C. The suspension illustrates a possible mechanism of this binding was centrifuged and washed twice with phosphate (531, 531a). Also illustrated is the participation buffer (0.05 M, pH 6.8). New glucan synthesis due to of S. mutans surface protein in the binding of cell- and glucan-associated GTase was measured by dextran (see next section). incorporation ofradioactivity from ['4CJsucrose. Ad- Cell-free water-soluble glucan "particles" herence to a glass surface was measured as percent- treated by sonic oscillation bind GTase and age of adherence of cell or glucan. Symbols: *-*, cell-bound '4C-labeled glucan synthesis; cause marked adherence to glass accompanied O-O, glucan-bound 14C-labeled glucan synthesis; by de novo glucan synthesis (Fig. 7; 237). This *-0, adherence of cells due to new glucan synthe- finding strongly supports the hypothesis that sis; 0-----0, adherence ofglucans due to new glucan GTases bound to the surface glucan participate synthesis. in the adherence of S. mutans cells to smooth surfaces when sucrose is present in the oral When S. mutans is grown in sucrose-free com- environment. Other water-insoluble glucans plex medium or the chemically defined medium such as amylopectin and cellulose do not bind FMC (575), the organisms do not have enough GTase significantly (238). cell-bound GTase to produce significant adher- 352 HAMADA AND SLADE MICROBIOL. ]REV. ence to solid surfaces when sucrose is added. On dium (623) show a markedly decreased ability to the other hand, cells grown in complex media agglutinate. Therefore, GTase associated with containing sucrose (e.g., Todd Hewitt broth and the surface glucan of the cell may augment the Trypticase soy broth) or sucrose-containing function of a "dextran receptor." FMC have strong cell-associated GTase activity. In this context, glucan/dextran-binding pro- These cells produce marked adherence to solid teins have been demonstrated in S. mutans. The surfaces in the presence of exogenous sucrose multiplicity of proteins showing this capacity or (232, 237). Furthermore, the presence of sucrose GTase activity indicates the complexity of the determines the ratio of cell-free and cell-bound cell-to-cell and cell-to-surface adherence mech- GTase (232, 237). anisms of S. mutans (178, 387, 497, 531). Kuramitsu (328) reported that a preformed In addition to cell-to-surface adherence de- glucan layer on a glass surface produced a partial scribed above, cell-to-cell adherence is of impor- adherence of heat-treated cells of two of four tance for dental plaque formation. Surface com- serotypes of S. mutans, claiming that glucan ponents which affect the aggregation ofbacterial synthesis need not be restricted to the cell sur- cells are therefore functionally critical for adhe- face of S. mutans for cellular adherence to de- sion among bacteria in dental plaque. velop. However, the presence of residual GTase Many strains of S. mutans agglutinate (ho- and sucrose in the precoated glucan has been mologous cell-cell adherence) upon addition of demonstrated, and the function of active GTase high-molecular-weight dextran (185). Certain in this case cannot be ruled out (215, 230, 314). strains of S. mutans are also reported to form An in vivo study in which conventional rats were aggregates with other bacterial cells such as used also supports the latter concept (598; see Nocardia and (heterologous cell-cell previous section). adherence) (188). Strains of A. naeslundii and Maltose has been demonstrated to inhibit the A. viscosus have been shown to form aggregates in vitro adherence ofS. mutans to glass surfaces more often with strains of S. sanguis and S. (11, 238, 439). Furthermore, the increased glucan mitior than with strains of S. mutans (77, 129, synthesis in the presence ofprimer water-soluble 188, 401). Coaggregation between A. viscosus dextrans inhibits adherence to a glass surface and S. sanguis is inhibited completely by,- (238). linked galactosides (i.e., lactose) (401). However, when S. mutans cells are coated with high-mo- Cell-to-Cell Adherence: lecular-weight dextran or grown in the presence Bacterial Aggregation of sucrose, they form visible aggregates with A. Cells ofS. mutans grown in a complex medium viscosus (23). have been shown to agglutinate upon addition Another example of heterologous cell-cell ag- ofhigh-molecular-weight dextran T2000 (molec- gregation is shown between S. mutans and Can- ular weight, 2 x 106) (185). This means that dida albicans. Artificial plaque formation by an whole cell agglutination is due to cells which are S. mutans strain is augmented when a C. albi- bound together by dextran molecules. The bind- cans strain is inoculated with the S. mutans ing of dextran has been demonstrated by using (416a). radioactive dextrans/glucans (554, 623). Agglu- Conversely, certain oral bacteria in plaque and tination of S. mutans strain 6715 (serotype g) is saliva are demonstrated to produce dextranase detected at pH 8.5 upon addition of 6 ng of that may inhibit the adherence of S. mutans to dextran T2000. This corresponds to about three smooth tooth surfaces (431, 504, 548, 603). molecules of dextran per cell in the reaction The increased synthesis ofpolysaccharides by mixture. plaque bacteria during a sucrose-rich diet is ac- Pretreatment of S. mutans cells with 4.0 M companied by increased levels ofdextranase and urea, 0.01 M EDTA, or 0.1% sodium dodecyl levanase of plaque bacteria (173). sulfate prevents agglutination, and divalent cat- It now appears from these various data that ions reverse the effect of EDTA (293). McCabe the adherence of S. mutans and other oral spe- and Smith (390) have reported that agglutina- cies to pellicle-covered teeth occurs in several tion is independent ofcell-bound GTase activity. steps. The initial attachment of single cells, GTase activity is almost completely abolished chains of celLs, or aggregated cells may involve by chemical treatments without adversely af- divalent ions (such as Ca2+) and the negative fecting the agglutination reaction. However, rab- charges on the bacterial cell and the tooth pel- bit antisera specific for GTase are shown to licle (486). This proposal, however, suffers from inhibit the agglutination reaction (449). Further- many limitations (181). It seems more likely that more, cells grown in sucrose-free complex me- a complex may form between a glycoprotein in dium (544) or chemically defined synthetic me- the pellicle and a polysaccharide on the bacterial VOL. 44, 1980 STREPTOCOCCUS MUTANS 353 surface. The reverse may also occur (181, 190, by infecting them with the phages or with free 233, 235). This may be a lectin-like effect (181). phage DNA. In addition, all transformants were The second phase of the process would depend reported to acquire a new character, the deami- in large measure on the multiplication of S. nation of arginine. Furthermore, S. sanguis mutans and glucan synthesis. The maturation of strain ATCC 10556 was transfected with free the plaque, containing various gram-positive and phage DNA of parent strain PK1, and two trans- occasionally gram-negative species, would be fectants which had developed the "cariogenic mediated by the synthesis of glucan by S. mu- nature" were obtained (256). However, the car- tans. Glucan would ensure the stability of the iogenicity of these strains was not tested. No plaque. Bacterial species which enter the devel- explanation was given of the relationship be- oping plaque after the phase of initial attach- tween prophage and plasmid DNA of parent ment may do so by random contact with the strain PK1. adhesive glucan of the plaque (233, 235). A small plasmid has been isolated from S. OF S. mutans strain LM7 (serotype e). The plasmid GENETIC ASPECTS MUTANS has a molecular weight of approximately 3 x 106 Lysogenicity and Plasmids and is calculated to have 16 copies per chromo- The occurrence of genetic elements such as somal genome equivalent (120). Since then, plasmid and prophage in bacteria may relate to more than 100 strains of S. mutans, including various phenotypes such as ability to ferment laboratory-maintained strains and clinical iso- sugars, production of toxins, bacteriocins, and lates, have been examined for plasmid DNA by antigens, and antibiotic resistance. examination of cell lysates on cesium chloride- In 1971, Greer et al. (200) reported that there ethidium bromide gradients (84, 377, 378, 471). was a consistent positive correlation between The frequency of occurrence of plasmid DNA in the lysogenicity and cariogenicity of S. mutans. S. mutans of human origin has been reported to "All" cariogenic streptococci including eight be approximately 5%. The value is lower than strains of S. mutans and one strain of S. sali- that of plasmids in other bacterial species, in- varius underwent lysis after induction with ul- cluding gram-positive and -negative species traviolet light and mitomycin C. In contrast to (145). cariogenic strains, "no" noncariogenic strepto- Macrina et al. (377, 378) have reported that coccal strains displayed the induced lysis. An- the four plasmids isolated by them are identical other group (309) also reported that a phage in molecular weight (3.6 x 106) and are present with similar morphology was induced from 15 to the extent of approximately 30 copies per cariogenic strains including A. viscosus and S. chromosomal equivalent. These results are es- sanguis in addition to 9 strains of S. mutans. sentially similar to those obtained with LM7 However, an S. mutans that had been cured plasmids obtained by Dunny et al. (120). In spite of its prophage exhibited cariogenicity essen- oftheir physiological comparison ofthe plasmid- tially similar to that ofits lysogenic parent strain containing and plasmidless S. mutans strains, when examined in animal models (R. J. Fitzger- they failed to find any clues regarding possible ald, personal communication). function of the plasmids, although production of Higuchi et al. (254) found that plasmid-curing bacteriocin-like activity was different in the two agents induced mutants at high frequency; these plasmid-containing strains. mutants produced diminished insoluble polysac- The few occurrences of plasmids in S. mutans charide. A satellite band of plasmid DNA in cell argue against earlier claims of plasmid-mediated lysates of parent strains of PK1 and JC2 was polysaccharide synthesis by this bacterium. Re- subsequently found, whereas mutants of these lated to this, Donkersloot et al. (112) isolated strains, that had lost the ability to synthesize mutants of S. mutans LM7 that had essentially adherent, insoluble polysaccharides had no de- no GTase activity, but still retained plasmid tectable satellite band of DNA (253). DNA. Therefore, these mutants are different Other investigators have been unable to find from the plasmidless, GTase-deficient mutants a plasmid in many strains of S. mutans, includ- of strains PK1 and JC2 (253). Furthermore, no ing strains PK1 and JC2 from which Higuchi et difference between the mitomycin C-induced al. (253) isolated plasmids. Furthermore, no phe- lysis of parent and mutant LM7 cultures was notypic function has yet been ascribed to any of observed, which is contrary to previous findings the reported plasmids of S. mutans. (254, 309). Similar results have been obtained Later studies reported (225) that only the with a "cariogenic" S. faecalis strain ND539. parent strain of PK1 (254) carried prophage and Isogenic pairs with or without plasmid that the mutant strains of PK1 were transformed (pAM539) are found to exhibit only a marginal to the "cariogenic" strain with adherence ability degree of caries activity (84), indicating that 354 HAMADA AND SLADE MICROBIOL. REV. these plasmids do not control cariogenic poten- competent for DNA from strain Challis (251). tial of these S. mutans strains. In certain bacte- A later study reported that DNA prepared rial species, the loss of virulence factor has been from a spontaneous rough mutant, G26-R, of S. demonstrated to be not necessarily accompanied sanguis transforms wild-type smooth strains by the loss of a plasmid (541a). G26-S, G30-S, and Challis-S of S. sanguis into It should be added here that Katayama et al. variants with rough colonial morphology. The (289) isolated a plasmid from seven "mucoid" rough S. sanguis G26-R strain shows an in- strains of S. mutans, using an enzyme, mutano- creased ability to adhere to solid surfaces in lysin, with lytic activity against the cell wall. vitro. However, the transformation with DNA They feel that use of mutanolysin results in a from S. mutans strain Ingbritt that produces a more consistent detection of the plasmid. rough colonial morphology was not successful The transfer of genetic elements by conjuga- (610). tion is well known in gram-negative bacteria. LeBlanc et al. (337) have recently reported that BACTERIOCINS OF S. MUTANS: a ,/ plasmid from a group F streptococcus, which MUTACINS codes for resistance to erythromycin and linco- Orginally, the term "bacteriocin" was applied mycin, is transferred to S. mutans, S. sanguis, to proteins of the colicin type which cause death and S. salivarius by cell-to-cell conjugal transfer of the bacterial host. These proteins had activity when donor and recipient cells are incubated on against strains of the same or closely related a membrane filter but not in broth cultures. bacterial species, and were adsorbed to specific The presence of the ,B plasmid in S. mutans receptors (470, 560). However, the activity spec- confers on this bacterium the ability to serve as tra of bacteriocins produced by gram-positive a /3-plasmid donor to other S. mutans strains bacteria, including S. mutans, are broader than with more than 50-fold-higher frequency than is those in gram-negative bacteria, and only a few obtained in the original transfer from group F bacteriocins ofgram-positive bacteria are known streptococcus to S. mutans. to have the properties of the colicins (560). Transformation Bacteriocinogeny Among S. mutans Transformation has been shown to occur Kelstrup and Gibbons (296) first reported that among most groups of streptococci and S. san- in stab culture using Typticase agar plates sev- guis (group H, strain Challis) (460). Davidson et eral S. mutans strains out of 13 oral streptococci al. (101) have shown that strain Challis is capa- tested produced inhibition zones against other ble of incorporating DNA prepared from strep- streptococcal strains, including those of S. py- tomycin-resistant strains of S. mutans, S. san- ogenes and enterococci, but not against unre- guis, and S. salivarius. However, transfer of lated bacteria such as lactobacilli, staphylococci, other genetic markers such as fermentation of and Escherichia coli. Because the inhibition sorbitol and mannitol and the synthesis ofwater- zones were not infective, the possibility of the soluble polysaccharide was not demonstrated. involvement of bacteriophages was excluded. All Reciprocal transformation is observed only be- bacteriocins were protease sensitive and had a tween SM resistant strain Challis and S. sanguis relatively low molecular weight. However, no strains ATCC 10556 and ATCC 10557, but not activity was demonstrated in broth cultures. between strain Challis and S. mutans (101). These bacteriocins required a stabilizing agent Subsequently, Westergren and Emilson (611) such as agar, agarose, starch, dextran, or glyc- examined the prevalence of competent strains erol. among oral streptococcal isolates and surveyed In subsequent studies, some additional strains the ability for transformation ofsome competent of S. mutans were found to produce bacteriocins strains when exposed to heterologous DNA. active against several species (298, 476, 624). They confirm that DNAs from streptomycin- Hamada and Ooshima (226), using the stab resistant strains offive oral streptococcal species culture method, found that some ofthe reference including S. mutans transform S. sanguis strain and freshly isolated S. mutans strains inhibited Challis. None of the S. mutans strains is trans- the growth of a wide variety of gram-positive formed to streptomycin resistance after expo- bacteria, including mycobacteria, streptomyces, sure to S. mutans DNA. In contrast to S. mu- and actinomyces. They proposed that the bac- tans, 13 out of 50 S. sanguis strains are found to teriocin of S. mutans be designated "mutacin" be competent in response to DNA from strep- to differentiate it from those produced by other tomycin-resistant S. sanguis 804 (611), whereas streptococcal species. The name has been ac- most spreading and twitching strains of S. san- cepted (46, 560). guis from the human throat are reported to be Among 113 clinical isolates of S. mutans from VOL. 44, 1980 STREPTOCOCCUS MUTANS 355 Japanese children (220), 84 strains (74%) in- 20,000, and it is sensitive to trypsin and pronase. hibited at least one of the ten indicator strains, It is lethal for various streptococcal strains be- which included S. mutans, S. salivarius, S. san- longing to Lancefield groups A, C, D, G, L, and guis, and S.pyogenes (225). This result indicated 0, but inactive against S. mutans strains of the high level of bacteriocinogenic ability of S. serotypes a, b, c, and d. The mutacin binds to mutans as compared with other gram-positive the sensitive cells as well as to the cells resistant bacteria (560, 605). Serotype c strains account to its lethal action. Recently, Perry and Slade for 85 of the 113 clinical isolates, and these (461) isolated the specific receptor molecule for strains produce mutacins more frequently than the mutacin from an S. pyogenes strain which those of other serotypes (225). Essentially simi- inhibited the activity of mutacin GS5. The re- lar results have been obtained by others (480). ceptor has a molecular weight of about 93,000 Very few strains are found to produce muta- and may be a heat-sensitive glycoprotein. cins extracellularly (225). Stabilizing agents did Delisle (102) obtained a bactericidal substance not support mutacin production (298). Mutacins from S. mutans strain BHT (serotype b) by are generally heat stable, and some are protease sonication or agitation of the culture with glass sensitive. Mutacins contain at least two kinds of beads. The chemical properties of this mutacin active components which have different molec- have not been described. The extracellular glu- ular weights (225, 479). Production of mutacins cans produced from sucrose by this strain did is influenced by the culture media used. Fur- not prevent mutacin production. Furthermore, thermore, when the indicator S. mutans strains sensitivity of the indicator strains producing are cultured in broth containing 5% sucrose, polysaccharides from sucrose remained unal- sensitivity to the mutacin decreases remarkably tered even when sucrose was present (103), con- (225). A coating of extracellular glucan most trary to the results of Hamada & Ooshima (225) likely renders normally susceptible organisms and Rogers (479). resistant to mutacin action (477). The only information available on the mech- Strains of S. mutans have been characterized anism of action of bacteriocins of streptococci and differentiated by the production of, and indicates an inhibition of DNA, RNA, and pro- sensitivity to, mutacins. Mutacin typing may be tein synthesis (512). No involvement of a plas- a useful tool in epidemiological studies (14, 298, mid in mutacinogeny has been reported, al- 478, 480). The possibility of maternal and/or though extensive surveys have been made (102, intrafamilial transfer ofS. mutans has been sug- 225, 226, 290, 294). gested based on the similarity of mutacin pat- More recently, S. Hamada and H. Imanishi terns (14, 478, 382). Rogers (478) reported that (unpublished data) found that a clinical isolate one mutacin type of S. mutans predominates in of S. mutans (serotype g) produced mutacin the individual human mouth. However, plural extracellularly in tryptose phosphate broth. Nei- serotypes of S. mutans were isolated from a ther yeast extract nor serum was required for single human subject (220, 382). These strains the production of the mutacin. It was heat sta- should have different mutacin patterns. ble, protease insensitive, and active against some Eleven of 17 human strains and 7 of 16 rat other S. mutans strains as well as other strep- strains of S. mutans, all of which are nonlyso- tococcal species. Similar mutacinogenic serotype genic, produce mutacins. Most of the nonmuta- g strains were isolated from the sister and cinogenic rat strains are tetracycline resistant mother ofthe patient, indicating an intrafamilial (290). transmission, as has been suggested by others (316). Characterization of this mutacin is now in Extracellular Mutacins progress. Although many strains of S. mutans produce A possible in vivo role for mutacins has been mutacins on solid agar plates, only a few strains suggested recently. The streptococcal species have been reported to produce mutacins extra- killed by a mutacin in vitro were also sensitive cellularly in broth culture of the same composi- to the mutacin in vivo (604). Rogers et al. (481) tion (225,294,458,479). Addition ofyeast extract reported that a mutacinogenic S. mutans strain (2 to 4%, final concentration) to Trypticase soy prevented the oral establishment of A. viscosus broth promoted the synthesis of extracellular Nyl when introduced into gnotobiotic rats. mutacins (225, 294). However, a nonmutacinogenic S. mutans strain Paul and Slade (458) isolated and partially did not show such an inhibitory effect. Similar purified the extracellular mutacin from S. mu- findings have been obtained with a mutacino- tans GS5 (serotype c). It was necessary to add genic parent strain and a nonmutacinogenic mu- horse serum (5%, vol/vol) to the broth media to tant strain of S. mutans (482). Mutacin produc- obtain consistent activity. The mutacin GS5 is tion by S. mutans strains has been shown to a protein with a molecular weight of about occur in vivo, and this ability appears to be 356 HAMADA AND SLADE MICROBIOL. REV.

ecologically advantageous to the invading S. mu- surfaces (230, 427, 428, 447). The adherence in- tans strain in a microenvironment (585). Fur- hibition was found to depend on IgG antibody thermore, a cell-free preparation of mutacin (447). The antibody specific for the a-d site of from a serotype c strain was shown to inhibit the serotype a S. mutans was reported to inhibit caries induction by a mutacin-sensitive S. mu- the binding of GTase and subsequent adherence tans strain (T. Ikeda et al., Int. Assoc. Dent. of S. mutans cells (427). Antibody specific for Res. Abstr. no. 1147, 1979). These results indi- serotype e, but not cross-reacting group E anti- cate that some mutacins are concemed in the gen, inhibited adherence ofserotype e S. mutans ecology of the oral flora. cells. However, these antibodies did not prevent the binding of GTase to serotype e S. mutans IMMUNOLOGICAL ASPECTS OF (230). Similar findings have been obtained with S. MUTANS serotype d S. mutans cells (237). Distribution of S. mutans Serotypes Some antisera against whole cells ofS. mutans in Humans significantly inhibit the enzymatic activity of extracellular GTase and hence the subsequent It is essential to know the distribution of S. adherence to smooth surfaces (139, 222, 230, 427, mutans serotypes in human populations before 428, 538). Anti-GTase activity is not related to considering the immunological aspects of S. mu- whole cell agglutination titers (222), and the tans and dental caries. After the existence offive inhibitory activity cannot be diminished by ad- serotypes in S. mutans was established (32), the sorption with the homologous whole cells (139, geographical distribution of S. mutans serotypes 222). The inhibition by antiserum is serotype in plaque samples obtained worldwide was sur- dependent. Serotype c, e, and f and type a, d, veyed (32). Serotypes c and d were found in and g S. mutans are separated into two major every area. Serotypes a and b were also detected groups. Similar relations have been reported in samples from 6 and 9 areas, respectively, of with the antisera directed against extracellular the 14 studied. All of the plaque sampled from GTase of S. mutans (236, 331, 332, 538). American boys (14 to 16 years of age) contained Information on the penetration of antibody serotype a and d strains. Serotypes b and c were into the plaque is limited. In vitro S. mutans present in 74 and 8% ofthe samples, respectively, plaque was found to contain the specific anti- and no serotype e was detected (201). body at the plaque surface as shown by immu- However, later studies with isolates cultured nofluorescence (279). Additional studies are from human plaques have revealed that serotype needed. c is the most frequently detected serotype of S. Opsonization ofS. mutans, followed by phago- mutans, irrespective of age, country, sampling cytosis and killing by polymorphonuclear leu- site, or isolation and serotyping procedures. Se- kocytes, has been demonstrated by using anti- rotype c usually comprises about 80% of the total sera from rhesus monkeys immunized with isolates (38, 220, 368, 382, 459, 467, 577). whole cells of serotype c S. mutans (516, 517). Other as serotypes such d, e, f, and g have been The monkey antiserum to serotype c S. mutans occasionally isolated. However, it is surprising induced maximum phagocytosis and killing of that almost none of a serotype and b strains serotype c and e strains, which are immunolog- were found in most of the recent studies. This finding is in sharp contrast to the earlier reports ically related. Serotype a and d cells were also opsonized but to a lesser degree. demonstrating the prevalence of serotypes a and b in plaque samples (32, 201, 275). Other investigators also demonstrated, using Immunological Responses of Host to a biotyping method, that strains similar to se- S. mutans rotype c predominated (174, 292, 521, 522), al- Various antibodies reacting with S. mutans though the biotyping method cannot differen- have been detected in serum, saliva, and colos- tiate serotypes c and e (221, 577). trum by several investigators using different In Vitro methods. In many infectious diseases, serum Effects of Antisera Against antibodies play a protective role. Patients with S. mutans immunoglobulin dysfunctions have been found Antibodies raised against various cellular and to have a greater susceptibility to dental caries extracellular components of S. mutans have (87). In addition to humoral antibody responses, been shown to exert a variety of effects on the local immunity may be enhanced to contribute biological activities of S. mutans. Antisera to protection against diseases of mucosal sur- against whole cells of S. mutans markedly in- faces including the oral cavity (397). hibit the adherence of homologous or immuno- Only a slight difference in serum antibody logically related cells of S. mutans to smooth titers was observed between caries-free and VOL. 44, 1980 STREPTOCOCCUS MUTANS 357 rampant-caries groups (299). Cell-wall aggluti- the protective effect (47, 343, 344), whereas nation tests were used to detect antibody against American workers suggest that secretory IgA in S. mutans in sera. However, the cell walls were saliva inhibits adherence of S. mutans to tooth prepared from serotype a, b, and d S. mutans, surfaces (138, 400, 409, 573). However, it should which were later found to be rare serotypes in be noted that these two mechanisms are not the human population as discussed above. More necessarily mutually exclusive. recently, it has been suggested that the immu- A preliminary study has shown that three irus nological response against dental caries is asso- monkeys vaccinated with whole, live cells of a ciated with the proportion of IgG to IgA and serotype c S. mutans developed significantly IgM classes ofantibodies to serotype c S. mutans fewer carious lesions than control, nontreated (346). monkeys (24). Subsequently, it was demon- Many oral bacterial species have been found strated that whole cell or broken cell vaccines to react with salivary antibody (29). Significant conferred significant protection in monkeys, es- levels of agglutinins specific for the five sero- pecially when the immunogen was administered types of S. mutans were detected in normal by intraoral submucosal injection (26). It was human colostrum and saliva, whereas relatively suggested that the induction of local immunity low levels were found in serum. The agglutinin is not a prerequisite, because good protection activity was identified as secretory IgA (5). It is was obtained by immunization via both submu- suggested that antigenic stimulation occurs at a cosal and subcutaneous routes (85). Protection site remote from the oral cavity, because secre- was not obtained with glucosyltransferase prep- tory IgA to the bacteria of the indigenous oral arations in the monkey test system (26, 85). flora was found in the colostrum as well as the Lehner et al. (342, 343) have reported the saliva (398, 414). immunization of rhesus monkeys with serotype Arnold et al. (4) further demonstrated that 8 c S. mutans cells mixed with incomplete Freund of 25 patients with selective IgA deficiency had adjuvant. They found protection with use of the significant levels of IgM in their saliva. In the complete immunogen and a delayed onset of normal control group, IgA was responsible for caries with the adjuvant alone. Immunized ani- antibody activity. These results suggest a biolog- mals contained demonstrable serum antibody to ical activity for secretory IgM which compen- GTase that inhibited GTase activity (498). Im- sates for the absence of secretory IgA. munization enhanced serum IgG and IgM titers, A positive correlation between increased car- whereas there was little increase in the salivary ies incidence and decreased levels of salivary IgA titers in the immunized compared with non- IgA in humans has been reported by different treated control monkeys (340). The reduction in groups of investigators (41, 66, 140, 141, 341, caries was associated with a reduction in the 453). Recently a significant negative correlation number of CFU of S. mutans in the fissures has been reported between salivary IgA specific (340). A correlation was found between the CFU for serotype b S. mutans and the dental caries of S. mutans and the number of carious lesions of 20 children (3 to 7 years old), implying that in rhesus monkeys (47). More recently, protec- IgA provides protection against dental caries tion was demonstrated against dental caries in (142). rhesus monkeys infused passively by the intra- IgA antibodies reacting with serotype c S. venous route with antibodies of the IgG class. mutans in secretions from minor salivary glands Intact molecules of IgG, IgA, and IgM have been of humans have been found (234). Furthermore, shown to pass from plasma to the oral cavity via parotid saliva from all subjects tested had IgA crevicular fluid, and therefore can contribute to antibodies to various serotype-specific polysac- local defense mechanisms (67, 343). charides, LTAs, and the peptidoglycan of S. In contrast to the findings described above, mutans (33-35). It should be noted that a signif- ample evidence has been reported indicating the icant variation of antibody titers was observed primary importance of local immunity due to during the experimental periods (33, 35, 234). secretory IgA antibodies. Taubman and Smith (573) demonstrated that local immunization Possible Vaccination with with formalinized whole cells of S. mutans re- S. mutans Antigens sulted in an enhanced salivary IgA response and Immunization with S. mutans is an attractive reduced caries development in both conven- concept for the control of dental caries (339). In tional and gnotobiotic rats. It was also found this respect, two different hypotheses have been that similar immunization using cell-free GTase proposed for the mechanisms of immunological preparations with Freund complete adjuvant re- control against dental caries. One hypothesis, sulted in the presence of antibodies in saliva of put forward primarily by British groups, is that rodents (540, 574) and monkeys (7). Oral im- serum IgG antibodies are mainly responsible for munization of hamsters with the enzyme pro- 358 HAMADA AND SLADE MICROBIOL. REV. duced similar results (540). Reductions in car- adsorbed from pooled acute sera ious lesions were greater on smooth surfaces of by S. mutans 6715 (587). The quantity of anti- teeth than on occlusal surfaces, probably due to body necessary to reduce the caries rate in hu- interference with adherence of S. mutans (325, mans after oral immunization with GTase may 574). In these cases, salivary antibody is the not be detrimental to the host (540). most likely protective principle in the rodents. Other antigens of S. mutans deserve consid- Local immunization with whole cells of S. eration as a vaccine to reduce the incidence of mutans stimulates a specific salivary IgA re- dental caries. Antibodies to the type-specific sponse which is protective against caries induc- polysaccharide and the glucan of serotype a tion by S. mutans (400). Similar find- (strain HS6) will inhibit the in vitro adherence ings have been demonstrated in irus monkeys of this species (427, 428). Type-specific polysac- by injection of S. mutans into the vicinity of the charide antibodies to the type e (strain MT703) major salivary glands or parotid ducts (134, 138). have the same property (231). The isolation of The ingestion of Formalin-treated cells of a a rhamnose-rich polysaccharide from the cell serotype g S. mutans strain has been shown to wall of serotype d S. mutans (462) indicates that stimulate specific secretory antibodies in saliva polymers other than the serotype polysaccha- and milk but not in serum of rats. These anti- rides remain to be characterized. Polysaccha- bodies were found to be of the IgA class. Orally rides would be expected to produce fewer reac- immunized rats developed significantly fewer tions when used as an antigen in humans. carious lesions than nontreated control rats (409). The level of specific salivary IgA antibod- CARIOGENICITY OF S. MUTANS ies in rats correlated with a reduction in the IN EXPERIMENTAL ANIMALS level of plaque and caries scores and the viable counts of S. mutans in plaque (408). Caries Induction in Animals Intravenous or intramucosal administration of After the epoch-making experiments by Or- vaccines to female rats produced elevated IgG land and his co-workers (451) in which germfree in colostrum, milk, and serum and elevated IgA rats were used, Fitzgerald and Keyes (157) dem- in colostrum and milk, respectively. Passive onstrated in 1960 that certain streptococcal transfer of either IgG or IgA has been found to strains isolated from carious lesions of rats and render protection against caries development in hamsters could produce caries in gnotobiotic rat offspring (399, 407). These results support rats and "caries-inactive" hamsters (156, 157, earlier findings (345). 302). These strains are not termed "S. mutans." A recent report (406) has indicated that a The "caries-inactive" hamsters have been found secretory IgA immune response is elicited in to be free from indigenous microflora which humans by ingestion of capsules that contain could induce dental caries when a caries-induc- Formalin-treated cells of S. mutans strain ing high-sucrose diet is fed. Once S. mutans is OMZ176 (serotype d). No increase in serum established in the mouth of the animal, caries antibody levels was demonstrated. It is of inter- activity is transmitted from parent to offspring est to note the simultaneous appearance of an- (157, 302). tibodies in remote secretory glands such as the In the earlier stage of caries research, it was salivary and lacrymal glands without a serum thought that there might be a specificity be- antibody response. tween the caries-inducing streptococci and the Protein-malnourished rats exhibited in- host animal species. However, Zinner et al. (628) creased caries susceptibility (405). However, a demonstrated that human strains of S. mutans, nutritionally compromised rat can elicit a spe- which reacted with the antiserum against the cific immune response that protects against S. hamster strains of S. mutans, could produce mutans-induced caries (411). extensive caries in hamsters. Since then, many It should be noted here that immunological streptococcal strains isolated from the human cross-reactions have been observed occasionally mouth have been shown to be cariogenic in between human heart tissue and certain com- various animal model systems (126, 127, 151, ponents of S. mutans strains (85, 498, 587). 183, 202, 207, 229, 321, 322, 433). Most of the These antigens have not been described, but cariogenic strains belong to the species S. mu- their presence in vaccine antigens is of great tans. However, organisms other than S. mutans concern. It seems possible, however, that certain can occasionally induce variable levels of caries antigens, GTase for example, may be useful in animals (for review, see reference 196). (540). Low concentrations of IgG antibody to Dental caries have been induced in various this enzyme exist in the serum of young normal kinds of animals, including monkeys (24), gerbils adults (22a). Heart-reactive antibody was not (150), mice (229, 551), rats, and hamsters. The VOL. 44, 1980 STREPTOCOCCUS MUTANS 359 transmission ofS. mutans from hamsters to mice with the fact that cells of these mutant strains and caries induction in mice have also been bound significantly lower quantities of extracel- demonstrated (551). lular GTase from S. mutans (235). Strains of S. mutans, regardless of their sero- Furthermore, the mutants differed from the types, almost always induce smooth-surface and parent strain in that each failed to form plaque pit-and-fissure caries in animals (229, 322, 410). on the smooth surface of the teeth and to pro- Strains of serotypes a, d, and g S. mutans tend duce smooth caries in specific-pathogen-free and to produce smooth-surface caries preferentially gnotobiotic rats (567). However, these mutants in rats (229, 410). However, variations are fre- produced sulcal caries, although with diminished quently observed in the pattern and severity of intensity. These results indicate that surface- the induced carious lesions in experimental ani- associated glucan synthesis by S. mutans appar- mals (366). In general, young animals are more ently contributes to the local environment and susceptible to a caries attack (335, 412, 434, 515). promotes the pathogenic potential of S. mutans Dietary factors critically influence the com- on smooth tooth surfaces. This is probably due position and pathogenic potential of inoculated to a barrier effect of the glucan layer to the S. mutans by affecting the implantation, colo- diffusion of metabolically excreted lactic acid, nization, and metabolic activities of the bacte- which has been considered to be critical in the rium. Sucrose has been demonstrated to be most demineralization of the teeth (567). The above cariogenic and supports the most rapidly pro- results also indicate that cell-to-surface adher- gressive pathogenesis, although other sugars, ence via insoluble glucan synthesis from sucrose such as maltose, lactose, and fructose, also sup- is a more important factor than cell-to-cell ag- port the induction of dental caries in animals to glutination induced by glucan in the pathogen- some extent (51, 168, 563). esis of dental caries. A surface fuzzy coat is suspected to contain a Noncariogenic and Supercariogenic glucan receptor which may be responsible for Mutants of S. mutans glucan-induced agglutination in both parent and To identify a virulence factor in the pathogen- mutant strains of S. mutans 6715. On the other esis of an infectious disease, mutants which lack hand, only the parent strain produces extracel- one or more characteristic properties possibly lular glucans with predominantly fibrillar mor- responsible for pathogenic processes are a useful phology (432). tool to analyze the mechanism of the pathogen- Other investigators also isolated similar types esis. of mutants from serotype g S. mutans strain In the work cited below, it should be noted AHT (308, 314) and serotype c strains GS5 (28, that the presence of a single mutation has not 280, 281) and PS14 (415). A mutant of S. mutans been established. It is reasonable to assume, LM7 (serotype e) forming little cell-bound glu- based on the techniques used, that more than can has been reported to attach to the teeth of one mutation is present. Also, a mutation, if rats comparably to its parent. However, cari- present, may be only indirectly related to the ogenic activity ofthese strains was not compared character(s) being considered. (79). De Stoppelaar et al. (107) isolated a mutant Mutants which produce elevated levels of which failed to synthesize cell-bound glucan in GTase have been isolated independently by two 5% sucrose broth from a serotype c strain of S. different groups (415, 502). These mutants dem- mutans. The inability to synthesize insoluble onstrate increased ability to adhere to glass sur- glucans of an adherent nature was accompanied faces (415, 502) and produce more carious lesions by a significant reduction of cariogenic potential than the parent strain (415). Thus, a clear cor- in experimental animals. The mutant also relation has been demonstrated between cari- showed a dramatic loss of viability due to acid ogenicity, in vitro adherence, and insoluble glu- production from either glucose or sucrose (113). can synthesis in S. mutans. Freedman and Tanzer (165) isolated mutants Other types of mutants that synthesize or ofS. mutans 6715 (serotype g) that differed from degrade less IPS have been isolated from two each other in colonial morphology on MS agar. serotype c strains which are strong producers of They found that the mutants lost the ability to IPS (166). These mutants had diminished viru- adhere to a wire surface but retained the ability lence both on smooth tooth surfaces and in to agglutinate and form macroscopically visible fissures (568). The loss of cariogenicity of these clumps in the presence of sucrose or exogenous mutants is attributed to diminished ability to glucans. The mutants were found to produce produce acid from endogenous IPS storage in increased amounts ofwater-soluble extracellular the absence of exogenous carbohydrates. How- glucans (163, 165). The latter finding coincides ever, strains of serotype dlg which have low 360 HAMADA AND SLADE MICROBIOL. REV. IPS-forming ability (164) do not appear to de- ies which relate the microbes of the carious pend on this property for caries induction as do lesion or dental plaque to the initiation of caries strains of serotype c. at the tooth site. The rationale for the hypoth- esis that S. mutans is strongly associated with S. MUTANS AND DENTAL CARIES human caries has been supported by the follow- IN HUMANS ing epidemiological studies. Effect of Sucrose on the Proportion S. mutans was isolated from all carious lesions, of S. mutans whereas only 23% of the samples from sound tooth surfaces of children (13 to 14 years old) The famous Vipeholm study provided strong contained the bacterium (362). Similar tenden- support for a close relationship between sucrose cies were also found in younger (135, 553) and intake and human caries prevalence (213). Re- older (17 to 22 years old) (257, 523) subjects. cent studies with gnotobiotic rats (413) have In an extensive study, it was concluded that revealed that as little as 0.1% sucrose in the diet there is a strong association between percentage can significantly promote the development of levels of S. mutans in single occlusal fissures and dental caries by S. mutans 6715, indicating that dental caries. Seventy-one percent ofthe carious the consumption of artificially high levels of fissures retained S. mutans, accounting for more sucrose is not necessary for the induction of than 10% of the viable count, whereas 70% of the dental caries. fissures free from caries had no detectable levels It is well known that dietary carbohydrates of S. mutans (372). Furthermore, it has been and infection with S. mutans are essential fac- shown that aciduric bacteria such as Lactoba- tors in the development of dental caries (303, cillus are detected in significant quantities in 511). Among dietary carbohydrates, sucrose is the dentinal carious lesion as the decay pro- considered to be directly related to dental caries gresses (373, 525, 561). (51, 168, 380). More recently, it was demonstrated that the Several studies on the effect of dietary sucrose proportion of S. mutans in samples from early on streptococcal composition in plaque flora carious lesions (white spots) of smooth tooth have been carried out with human subjects. surfaces was significantly higher than that from Carlsson and Egelberg (58) reported that plaque the adjacent sound surface. No significant num- formation was heavier during high-sucrose diet bers oflactobacilli were found in the early lesions periods than in glucose diet periods. When six (118). subjects were instructed to abstain from any However, the etiological involvement of a bac- dietary carbohydrates for 17 days, the S. mutans terium in the oral flora cannot be fully attributed count decreased to an undetectable level while by cross-sectional studies in the case of a chronic the percentage of S. sanguis increased (109). disease such as dental caries. To overcome the Such an inverse relationship between the S. problem, several longitudinal studies that dem- mutans and S. sanguis population was observed onstrate cause-and-effect relationships have in other investigations (167, 547). Other nutri- been reported. The distribution of S. mutans on tional interactions between S. mutans and S. the tooth surfaces was followed over a period of sanguis may be important for the ecology of 18 months. The development of caries was more these organisms in the oral flora (57). frequently preceded by colonization with ele- Contrary to an earlier study (58), it was re- vated levels of S. mutans (268). Subsequently, ported that high-sucrose diets had no significant other investigations (291, 310, 559) have led to effects on total plaque accumulation, although similar findings. tQtal viable microbial density and populations of On the other hand, no significant relationship S. mutans and lactobacilli increased (547). A between S. mutans and the initiation of dental low-sucrose diet did not completely eliminate S. caries in Danish preschool and British school mutans from the oral flora (547) as was shown children was found (246, 416). The variable re- in a study with monkeys (306). sults may be attributed to complex factors such as sampling sites, methods of cultivation, fluo- Epidemiological Relationship Between ride content, eating habits of the subjects, su- S. mutans and Caries Development crose intake, and possible immunity in the oral Many strains of S. mutans isolated from hu- cavity. mans have been demonstrated to be cariogenic It is of interest to note here that a significant in experimental animals as described above. increase in S. mutans in saliva and dental plaque However, these results do not necessarily apply is observed in patients who have received radia- to human dental caries. To clarify the etiological tion therapy of the major salivary gland. A close role of S. mutans in caries development in hu- relationship is established among rampant car- mans, we must depend on epidemiological stud- ies, xerostomia due to degeneration of salivary VOL. 44, 1980 STREPTOCOCCUS MUTANS 361 glands, and an increase in S. mutans (363). S. mutans has been reported to be highly In a survey of 22 infants over a period of 30 susceptible in in vitro tests with penicillin, am- months, no clear-cut association between the picillin, erythromycin, cephalothin, methicillin, development of caries and previous detection of and many other antibiotics (10, 147, 361). It is of S. mutans was reported (382). However, S. mu- interest that serotype a and b strains of S. mu- tans was isolated from all 12 of the infants who tans are very susceptible to bacitracin and po- developed caries. During the test period, lymixin B, respectively; other serotypes are not changes in the distribution of serotypes were susceptible (361). occasionally noted. Serotype dlg strains have a In spite of the in vitro effectiveness of anti- tendency to give rise to smooth-surface caries; biotics, it is not practical to use them for caries serotype c strains were always present (382). control. Recent investigations, however, suggest In this context, as the number oferupted teeth that certain antimicrobial agents may be used increases, there is a gradual increase in the prev- on a short-term basis to suppress S. mutans. alence of S. mutans. Edentoulous newborns or Such agents as vancomycin (105, 283), kanamy- aged men do not harbor significant quantities of cin (367), and iodine (64, 184, 570) can be used S. mutans (59, 62, 63, 382). It appears that S. topically for this purpose. mutans is most likely transmitted intrafamilially Other agents that have been reported to sup- (14, 15, 316, 382). press S. mutans and other cariogenic bacteria include fluoride (371, 374, 627), bisbiguanidines PREVENTION OF CARIES CAUSED BY (137, 197, 570), and surfactants (9, 571). Many of S. MUTANS these antimicrobial and antiplaque agents have In theory, dental caries can be prevented by been found to inhibit GTase activity of S. mu- eliminating cariogenic bacteria, especially S. mu- tans (74; Torii and Hamada, Abstr. Jpn. Assoc. tans, from the mouth, as well as by increasing Oral Biol., 1979). the resistance of teeth and modifying the diet Another unique method is the use of a bacte- (303, 305, 366, 437, 511). In the following sections, riolytic enzyme termed "mutanolysin," which emphasis has been paid mainly to methods in has been purified from a soil bacterium (625, the first category. 626). Most S. mutans strains, including labora- tory stock cultures and fresh clinical strains, are Suppression ofS. mutans by Antimicrobial markedly lysed by this enzyme (239). Mutano- Agents lysin inhibits the accumulation of dental plaque and the development of caries induced by S. The usefulness of penicillin in preventing ex- mutans strain AHT or KIR in specific-pathogen- perimentally induced caries has been noted since free hamsters (455). the pioneering work of McClure and Hewitt (396); they suspected Lactobacillus acidophilus as a causative agent. Since then, ample evidence Inhibition of Adherence of S. mutans by has accumulated which shows that most anti- Glucan-Hydrolyzing Enzymes biotics with antimicrobial activity against gram- The synthesis of insoluble adherent glucan positive bacteria depress the development of from sucrose by S. mutans is a prerequisite for dental caries induced in experimental animals the induction of dental caries in experimental (18, 152, 153). Furthermore, young human pa- animals. Plaque deposits on wire can be removed tients (6 to 19 years old) who had received long- by a dextranase preparation obtained from Pen- term administration of penicillin and/or tetra- icillium funiculosum (159). In vitro studies in- cycline for treatment of chronic infectious dis- dicate that dextranases, a(1-6) glucanases, eases developed about two-thirds fewer caries have limited ability to degrade the extracellular than did control subjects (242). This observation glucans produced by S. mutans (203, 224, 315, could be explained by the recent finding that the 435, 514). In subsequent studies, a(1-6) glucan- presence of the very low concentrations of both ases of different origins effectively prevented penicillin G and sulfadiazine markedly inhibits plaque formation and caries induction by S. in vitro plaque formation by S. mutans (607). mutans strains in hamsters (155, 159, 161). Sim- However, Weld and Sandham (608) reported ilar positive results have been obtained with that long-term therapy with penicillin and sul- other animal model systems (25, 227, 228, 463). fadiazine did not cause a significant reduction in Human clinical trials of a(l-.6) glucanases have the proportions of S. mutans or lactobacilli, al- resulted in conflicting antiplaque effects (48, 117, though the organisms isolated from the patients 199, 304, 364, 430). demonstrated high susceptibility to penicillin. Other investigators claimed that a(1-+6) glu- No penicillin-resistant strains of S. mutans have canase exerted no inhibition of plaque formation been described. or of caries induction in a rat test system (208). 362 HAMADA AND SLADE MICROBIOL. REV. They later reported that a(1- 3) glucanase, tween the isolates from human blood (54 strains) termed "mutanase," from a strain of Tricho- and dental plaque (50 strains) in terms of their derma harzianum (206) did inhibit caries induc- physiological characteristics (143). tion (210, 211). Preparations of a(l- 3) glucan- Recently, several investigators have con- ase have been reported to impair the coloniza- firmed that subacute bacterial endocarditis can tion of S. mutans in plaque (295) and to inhibit be caused by S. mutans (243, 260, 365). The the formation of dental plaque and gingivitis in patients had the typical picture of fever, heart humans (297). Several insoluble glucan-hydro- murmur, weakness, and repeated positive blood lyzing enzymes have been obtained from differ- cultures. Most patients had prior known valvular ent origins (123, 270). In vivo effects of these heart disease. Teeth were suspected to be the a(l,o3) glucanases have not been examined. locus of infection in some cases. All the strains More recently (527), a dextranase from a strain were susceptible to various antibiotics including of Fusarium moniliforme (528) had a much penicillin G (243, 260). All the patients were greater affinity for HA than did the Penicillium treated with penicillin G and streptomycin (243), dextranase. The investigators suggest the occur- but a fatal case did occur (365). rence of a more effective interference with the It is important that clinical laboratories dif- initial attachment of S. mutans and subsequent ferentiate S. mutans from the enterococcal spe- accumulation of dental plaque. cies. S. mutans is susceptible to low concentra- In this connection, yeast invertase that splits tions of penicillin G, in contrast to enterococci, sucrose into glucose and fructose is not regarded which are usually resistant to this antibiotic (10, as having a caries-inhibiting potential (160). Fur- 243). thermore, Gibbons and Keyes (186) reported Experimental endocarditis due to various bac- that addition of low-molecular-weight dextran teria can be readily induced in rabbits by placing into a caries-inducing diet inhibited plaque for- a catheter in the left side of the heart (121). mation and caries induction as well as the in With this model, Durack et al. (122) investigated vitro prevention of insoluble glucan synthesis by the effect of prior immunization with S. mutans S. mutans GTase. This finding, however, has and S. sanguis on the susceptibility of rabbits to not been supported by other investigators (520). experimentally induced streptococcal endocar- A "coupling sugar" preparation significantly re- ditis. Rabbits with a high complement-fixing duced caries activity in rats when it was substi- antibody titer to the infecting organisms devel- tuted for sucrose in a rat diet (269). The coupling oped the disease with a significantly lower fre- sugar is produced by incubating starch, sucrose, quency than those with lower titers. The results and cyclodextrin GTase from megate- do not support the concept that immunization rium (3, 446). with S. mutans for the prevention of dental caries may increase the susceptibility of the im- ENDOCARDITIS CAUSED BY munized subjects to an endocarditis caused by S. MUTANS S. mutans (122). Subacute endocarditis caused by streptococci The initial event of the pathogenesis of bac- is frequently due to the alpha-hemolytic and terial endocarditis is the attachment of bacteria nonhemolytic types. Abercrombie and Scott (1) to heart valves, particularly those with damaged first reported a case of endocarditis caused by a aortic valves possessing a platelet-fibrin throm- streptococcus that was considered identical to S. bus (2). Cell-bound glucan appears to promote mutans proposed by Clarke (81). In a recent the establishment of S. mutans and other glu- study in England, Parker and Ball (457) identi- can-producing streptococci on the heart valves fied the species of the 317 streptococcal strains (468, 510). Adherence to damaged valves is ap- which had been isolated from patients with sub- proximately five times higher than adherence to acute endocarditis. The most numerous are S. normal valves. These results may explain the sanguis (16.4%), S. bovis (15.1%), and S. mutans high prevalence of glucan-synthesizing strepto- (14.2%, 45 strains). It is of interest to note that cocci, including S. mutans, as the causative all of them as well as certain S. mitior strains agent of subacute endocarditis. Thus, the adher- (7.3%) synthesize glucan from sucrose. ence-promoting ability of glucan synthesized by Fifty-four strains of S. mutans from cases of S. mutans appears to be the initial step in the endocarditis in Denmark have been identified pathogenesis of endocarditis as well as dental and serotyped. The most prevalent was serotype caries. c (459). Seventy-seven stock strains isolated from human blood designated as S. bovis were SUMMARY rechecked, and 35 were identified as S. mutans It is likely that S. mutans is the primary cariogenic (106). There are no appreciable differences be- bacterium in both humans and animals. Other bacteria VOL. 44, 1980 STREPTOCOCCUS MUTANS 363 found in actively progressive carious lesions are con- Immun. 14:355-362. sidered to be secondary invaders, probably commensal 6. Bacon, J. S. D., B. Jones, V. C. Farmer, and with S. mutans with regard to their physiological D. M. Webley. 1968. The occurrence of activities. Only a limited number of species of bacteria a(l-*3) glucan in Cryptococcus, Schizosaccha- other than S. mutans are occasionally found to be romyces, and Polyporus species and its hy- cariogenic in experimental animals. drolysis by a Streptomyces culture filtrate lys- Virulence factors of S. mutans responsible for its ing cell walls of Cryptococcus. Biochim. Bio- cariogenicity include the ability to adhere to smooth phys. Acta 158:313-315. surfaces and acidogenic-aciduric properties. Adher- 7. Bahn, A. R., I. L. Shklair, and J. A. Hayashi. ence to smooth tooth surfaces is responsible for cari- 1977. Immunization with dextransucrases, lev- ogenic plaque formation by S. mutans and is mediated ansucrases, and glycosidic hydrolases from oral by the de novo synthesis of a glucose polymer from streptococci. II. Immunization with glucosyl- dietary sucrose. The synthesis is due to the action of transferases, fructosyltransferases, and glyco- cell-free or cell-associated forms of GTases. This ex- sidic hydrolases from oral streptococci in mon- plains the marked caries-inducing property of sucrose keys. J. Dent. Res. 56:1586-1598. in diets. 8. Baird, J., V. Longyear, and R. Ellwood. 1973. In terms of bacterial , the species S. mu- Water insoluble and soluble glucans produced tans includes a number of heterogeneous strains. Var- by extracellular glucosyl transferases from ious immunological, biological, and biochemical prop- Streptococcus mutans. Microbios 8:143-150. erties and the epidemiological distribution of S. mu- 9. Baker, P. J., R. A. Coburn, R. J. Genco, and tans have been discussed in this review with special R. T. Evans. 1978. The in vitro inhibition of reference to the seven serotypes ofthe microorganism. microbial growth and plaque formation by sur- The occurrence of S. mutans in subacute endocarditis factant drugs. J. Periodontal Res. 13:474-485. and the possibilities of a vaccine against dental caries 10. Baker, C. N., and C. Thornsberry. 1974. An- have also been discussed. timicrobial susceptibility of Streptococcus mu- tans isolated from patients with endocarditis. ACKNOWLEDGMENTS Antimicrob. Agents Chemother. 5:268-271. 11. Balekjian, A. Y., R. W. Longton, J. S. Cole We thank F. C. McIntire for his generous coopera- EII, and M. S. Guidry. 1977. The effect of tion in providing facilities in the Department of Oral disaccharides on the plaque-forming potential Biology during the writing of a large part of this of Streptococcus mutans. J. Dent. Res. 56: review. We are also grateful to Wanda Valentine and 1359-1363. Chiaki Kimura for excellent secretarial help, and to 12. Bammann, L. L., W. B. Clark, and R. J. Gib- Albert E. Vatter for the electron microscopic work. bons. 1978. Impaired colonization of gnoto- The funds to support S. H. during his stay in Denver biotic and conventional rats by streptomycin- were kindly provided to H.D.S. by the Pioneer Fund. resistant strains of Streptococcus mutans. In- The work reported here in the laboratory of H.D.S. at fect. Immun. 22:721-726. Northwestern University Medical-Dental Schools was 13. Beachey, E. H., G. L. Campbell, and I. Ofek. supported by the National Institute for Dental Re- 1974. Peptid digestion of streptococcal M pro- search, the National Institute for General Medical tein. II. Extraction of M antigen from group A Sciences, and the National Heart and Lung Institute streptococci with pepsin. Infect. Immun. 9: of the National Institutes of Health, Public Health 891-896. Service, and by the Pioneer Fund. 14. Berkowitz, R. J., and H. V. 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