Virulence of Streptococcus mutans: a Sensitive Method For

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1 INFEcriON AND IMMUNrry, July 1975, p Copyright American Society for Microbiology Vol. 12, No. 1 Printed in U.S.A. Virulence of Streptococcus mutans: a Sensitive Method For Evaluating Cariogenicity in Young Gnotobiotic Rats SUZANNE M. MICHALEK, JERRY R. McGHEE,* AND JUAN M. NAVIA Department of Microbiology* and Institute of Dental Research, University of Alabama in Birmingham, Birmingham, Alabama Received for publication 23 January 1975 Gnotobiotic rats infected with Streptococcus mutans 6715 at 19 days of age and fed a purified diet (305) containing 5% sucrose developed extensive caries lesions on all molar surfaces within 16 days (35 days of age). Approximately twice as many lesions developed when infected rats were maintained until 45 days of age, whereas noninfected rats did not develop caries when fed diet 305. Gnotobiotic rats infected with S. mutans 6715 and fed a purified diet containing no sucrose (300) until day 25 and subsequently fed diet 305 for 10 days developed lesions similar to rats fed diet 305 for 16 days. Furthermore, rats infected with S. mutans 6715 and fed diet 300 until 45 days of age developed approximately one-half the smooth surface lesions as infected rats fed diet 305 for the same length of time. The level of caries on buccal and proximal molar surfaces in 45-day-old gnotobiotic rats varied when animals were infected with S. mutans AHT, BHT, NCTC 10449, 6715, or LM-7. Animals infected with S. mutans AHT showed more severe lesions on the buccal surfaces than those observed in animals infected with the other strains of S. mutans tested, whereas S. mutans 6715 caused significantly more caries on proximal surfaces. On the other hand, rats infected with S. mutans LM-7 exhibited the lowest level of caries on all molar surfaces of the five strains of S. mutans tested. The first clear evidence that dental caries was high- and low-molecular-weight glucans (16, 20, a multifactorial, infectious disease of bacterial 37). These glucans form a coherent mass which etiology came from the studies of Orland and allows adherence of S. mutans to hard surfaces, co-workers (31,32). Their work demonstrated including tooth enamel, and thus contribute to that germfree rats did not develop caries even plaque formation (7, 12, 14-16, 19, 27). In when fed a diet which was caries promoting in addition to glucan synthesis, S. mutans ferments the hexose moieties of sucrose, with conventional rats (32). However, gnotobiotic rats infected with an enterococcus and fed the subsequent acid production (5, 6, 22). The acid same diet developed caries lesions (31). produced leads to decalcification of the enamel From these observations it could be concluded that in order to study experimental The germfree rat has been widely employed in surface with subsequent decay. dental caries three factors must be considered: studies to determine the caries-inducing potential of microorganisms (17). These studies have (i) the caries-promoting diet, (ii) the cariogenic microorganism, and (iii) the susceptible host. involved monoinfection with oral microorganisms and feeding caries-promoting diet for Purified caries-promoting diets have been developed (29); however, these contain unusually extended periods of time (9, 13). As in children, high concentrations of sucrose (56 to 67%). This, the young rat is more susceptible to caries of course, does not reflect the usual dietary attack, notably during the period of tooth intake of man. Nevertheless, recent work has maturation; this occurs within approximately confirmed the role of sucrose as the principle 14 to 21 days after weaning (25). substrate for oral cariogens, especially members The purpose of the present investigation was of the viridans streptococci, most notably (i) to determine the virulence of S. mutans 6715 Streptococcus mutans (10, 22, 23). in young gnotobiotic rats fed a purified cariespromoting diet (305) containing only 5% sucrose Numerous studies have demonstrated that S. mutans produces the enzyme glucosyltransferase, which in the presence of sucrose cariogenicity of five different S. mutans strains over short test periods and (ii) to compare the induces the synthesis of large amounts of both in this system. 69

2 70 MICHALEK, McGHEE, AND NAVIA MATERIALS AND METHODS Microorganisms and growth conditions. Five strains of S. mutans isolated from humans were employed in this study: AHT (38), BHT (38), NCTC (8), 6715 (11, 14), and LM-7 (13). These strains correspond to Bratthall's serological groups a, b, c, d, and e, respectively (2). Stock cultures of each strain were maintained at 4 C in brain heart infusion agar stabs containing excess CaCO3. For animal infectivity, cultures were grown under anaerobic conditions at 37 C in brain heart infusion broth. Log-phase cultures of each test organism were introduced into the appropriate isolator in tightened screw-capped tubes just prior to infection. Gnotobiotic animals and experimental design. Germfree Fischer rats [CD F(344)GN] employed in this study were originally obtained from Charles River Laboratories (Wilmington, Mass.). Rats were maintained in Trexler plastic isolators (36) and provided with sterile Purina rat chow and water ad libitum. Adult rats were mated and, after delivery, rat pups were pooled, randomized, and assigned seven to eight pups per dam. The overall experimental design employed in this study is presented in Fig. 1. Pups (19 days of age) were transferred to separate isolators prior to infection. Control pups were maintained in the original isolator. All pups were fed either diet 300 or 305 (described below). Infection was carried out by a single oral treatment of each pup (day 19) with a swab saturated with a log-phase culture (approximately 2 x 108 colonyforming units/ml) of the appropriate test S. mutans. Fecal samples and oral swabs were collected from individual animals on the following day and cultured in thioglycolate and brain heart infusion broth and on blood agar, Mitis-Salivarius agar, and Sabouraud agar plates. Cultures were incubated at 25 and 37 C under aerobic and anaerobic conditions to verify colonization of the infecting S. mutans and to insure absence of extraneous contamination. Continued colonization by the test S. mutans was verified by culture on Mitis-Salivarius agar at approximately weekly intervals. One hundred percent colonization of over 300 rats treated by this technique has been observed. Furthermore, the S. mutans which was isolated always demonstrated morphological and metabolic characteristics identical with the original test strain. Rats were removed from isolators at either 35 or 45 days of age, and fecal samples and oral swabs were collected and cultured (as described above). The test S. mutans was always isolated from each infected rat, and no other bacteria or fungi were detected in any of the experiments reported here. The group specificity of the isolated S. mutans was determined by sugar fermentation (8, 18) and serological testing with group-specific antisera (2). Rats were weighed and sacrificed with the aid of a guillotine, and the heads were autoclaved for 5 min to loosen soft tissue. The mandibles were dissected, cleaned, and then stained with 0.4% murexide in 70% alcohol. Caries-promoting diet. Purified diet 305, previously described by Menaker and Navia (28), was employed in this study (Table 1). Since the diet TABLE 1. Composition of purified, caries-promoting diet for young gnotobiotic rats Components INFECT. IMMUN. Purified diet 305a (%) Sucrose (6 x pulverized) Lactalbumin Salt mixture (MIT)b Vitamin mixturec Cottonseed oil Cellulose Cornstarch abased on purified diet 200 (30). Diet 300 substitutes cornstarch for sucrose. b MIT salt mix (grams per kilograms of mix): CaHPO4, ; KCl, ; NaCl, ; K2SO4, 61.83; MgSO4, 19.82; MgCO,, 27.28; iron citrate, 18.00; MnCO3, 3.63; CuCO3, 0.529; ZnCO3, 2.70; KI03, ; AlCl36H20, ; CoCl2-6H20, cvitamin mix (grams per kilograms of diet): thiamine, 0.008; riboflavin, 0.006; pyridoxine, 0.003; B12 (0.1%), 0.03; niacin, 0.02; calcium pantothenate, 0.03; p-aminobenzoic acid, 0.20; inositol, 2.00, biotin, ; menadione, 0.003; folic acid, 0.002; a-tocopherol, 0.4; ascorbic acid, 0.100; vitamin A acetate and D2 (Roche, 1:10), 0.04; choline HCl, 3.00; cellulose, This represents a double concentration of that used in conventional animal experiments. contains a low concentration of sucrose, sterilization was accomplished by autoclaving. Twice the original amount of vitamins was added to insure optimal nutritional quality. After mixing, 300 g of diet was dispensed into Brown-n-bags (10 by 16 inches [ca by 40.6 cm]; Reynolds Metal Co., Richmond, Va.). These bags were flattened and autoclaved together with stainless-steel metal cans (30 min at 121 C). After sterilization, the bags of diet were immediately placed into the metal cans and covered; upon cooling, the cans were treated with 6% peracetic acid and introduced into the isolators. Sterilization resulted in the diet assuming a pale brown color; however, the fine powder consistency was maintained. Nutritional adequacy of the autoclaved diet was determined by feeding one group of conventional weanling rats (COBS/CD, Charles River Laboratories) sterile diet 305 and a second group, nonsterile diet 305. Both groups of rats exhibited identical weight gains and overall good health. Purified diet 300 was employed in two series of experiments. This diet is identical to diet 305 except sucrose is replaced by cornstarch (Table 1). Caries scoring. After staining of mandibles with murexide (0.4%), molars were hemisectioned, and buccal, sulcal, and proximal surfaces were scored for caries by the Keyes procedure (21). In each experiment, the caries scores from each group of rats were statistically reduced by computing means, standard deviations, and standard errors. Differences among means were evaluated by analysis of variance and multiple mean comparisons using the Duncan test.

3 VOL. 12, 1975 RESULTS Studies with S. mutans In the first series of experiments, germfree rats were weaned (19 days of age), infected with S. mutans 6715, fed purified diet 305, and sacrificed at either 35 or 45 days of age (Fig. 1). Infected rats fed diet 305 for 16 days (group A) had extensive caries on buccal and sulcal surfaces and moderate proximal lesions (Table 2). Mean caries scores in infected rats fed diet 305 for 26 days (Table 2, group D) were approximately twofold higher than observed in 35-day-old infected rats. The severity of decay on all tooth surfaces in 45-day-old infected rats is illustrated in Fig. 2A, whereas noninfected rats fed diet 305 were caries free (Fig. 2B). Both infected and noninfected gnotobiotic rats exhibited similar weight gains (Table 2) and overall good health. These results clearly demonstrated that purified diet 305 is both nutritionally adequate and caries promoting when fed to young gnotobiotic GROUP Day 1 9 DO INFECTION VIRULENCE OF S. MUTANS IN GNOTOBIOTIC RATS rats infected with S. mutans The data presented for one experiment (Table 2) are representative of five separate studies. To further test the sensitivity of this model for caries induction, a second series of weanling, gnotobiotic rats were fed purified diet 300 (which contains no sucrose) until day 25, followed by diet 305 until 35 days of age. One group of rats was infected with S. mutans 6715 at 19 days of age (Fig. 1, group B), and the second group served as noninfected controls. Colonization of S. mutans 6715 was confirmed the next day andon day 25. After only 10 days of exposure to diet 305, the infected rats had extensive caries on buccal and sulcal surfaces and moderate proximal lesions (Table 2, group B). It was of interest, that the mean caries scored in this group of infected rats was not significantly different from scores obtained from 35-day-old infected rats fed diet 305 for 16 days (Table 2, group A). Furthermore, both groups of 35-day-old infected rats showed simi FED PURIFIED DIET 305 FED PURIFIED FED PURIFIED DIET 305 DIET 300 C FED PURIFIED DIET 300 D FED PURIFIED DIET 305 SACRIFICE SACRIFICE FIG. 1. Experimental design employed to evaluate the cariogenicity of S. mutans in gnotobiotic rats. TABLE 2. Mean caries scores in gnotobiotic rats infected with S. mutans 6715 Day Mean caries scoresa Group" sof sacri- Buccal Sulcal uclwtc Proximal Mean body fice E D. D. D. E D 71 A ± ± ± ± D ± ± ± ± ± ± ± 1.19 B i ± ± ± ± 0.55 j ± 1.56 a EValuated by the Keyes procedure (21); E, Penetration into enamel; D., slight penetration into dentin; D., extensive destruction of dentin. Score + standard error (represents 15[A], 23[D], and 10[B] rats). b Control (noninfected) germfree rats had no lesions on any surface at the times tested (total of 28 rats) (see Fig. 1). Groups A and D were fed diet 305; group B was fed diet 300 until day 25 followed by diet 305 for 10 days. c Expressed in grams + standard error; 35- and 45-day-old control, noninfected rat weights were within these standard errors.

4 72 MICHALEK, McGHEE, AND NAVIA INFECT. IMMUN. Downloaded from FIG. 2. (A) Lesions resulting from infection with S. mutans 6715 in a 45-day-old gnotobiotic rat fed diet 305. (B) Control, noninfected 45-day-old gnotobiotic rat fed diet 305. lar weight gains (Table 2). These findings suggest that (i) S. mutans can colonize germfree rats in the complete absence of sucrose, and (ii) 35-day-old, infected gnotobiotic rats had similar mean caries scores when fed diet 305 for either 10 or 16 days. In this regard, it was of interest that 45-dayold gnotobiotic rats infected with S. mutans 6715 and fed diet 300 continued to be infected by the organism throughout the 26-day period and developed some lesions on all smooth surfaces (Table 3). However, the smooth surface dentinal lesions were 2.7-fold (D8) and 9.6 fold (D.) lower on the buccal surfaces and 3.2-fold (D.) and 4.0-fold (Din) lower on the proximal surfaces as compared to scores observed in 45-day-old infected rats fed diet 305 for 26 days. Both groups of animals had similar weight gains (Table 3) and overall good health. From this series of experiments one can conclude that sucrose, even at a level of 5%,.is critical for development of rampant decay in 45-day-old gnotobiotic rats infected with virulent S. mutans. Studies with other S. mutans strains. Since young gnotobiotic rats infected with S. mutans on January 9, 2019 by guest

5 VOL. 12, 1975 VIRULENCE OF S. MUTANS IN GNOTOBIOTIC RATS TABLE 3. Mean smooth surface caries scores in gnotobiotic rats infected with S. mutans 6715, fed either diet 300 or 305 and sacrified at 45 days of age Mean smooth surface caries scores" Group' Buccal Proximal eac E D. D. E D. Dm C k k ± D t f f aevaluated by the Keyes procedure (21); E, penetration into enamel; D., slight penetration into dentin; D,,, moderate penetration into dentin; D., extensive destruction of dentin. Score + standard error. "As outlined in Fig. 1. c Expressed in grams k standard error; wveights of control, noninfected rats were within these standard errors developed rampant decay on all molar surfaces within 26 days when fed diet 305 (Table 2, group D), the virulence of four other strains of S. mutans was determined under identical experimental conditions (Fig. 1, group D) and compared with S. mutans 6715 (Fig. 3). No significant difference was observed in the level of enamel lesions on buccal surfaces in groups of rats (14 rats/group) infected with S. mutans AHT, BHT, 10449, or However, rats infected with S. mutans AHT had more dentinal lesions on buccal surfaces than were found in rats infected with any of the other strains tested. Buccal dentinal lesions resulting from infection with either S. mutans BHT or did not differ from each other but were 10 to 15% lower than those resulting from infection with S. mutans Rats infected with S. mutans 6715 had 33 to 55% higher enamel lesions and 25 to 50% higher dentinal lesions on proximal surfaces than rats infected with any of the other S. mutans tested. Of the five strains of S. mutans studied, LM-7 was the least virulent. DISCUSSION With the growing body of information on the pathogenesis of S. mutans infection, an increasing need exists to standardize procedures for in vivo evaluation of S. mutans virulence. Our study presents a rapid and sensitive model for the study of dental caries, thus allowing for more experiments to be performed. Reproducible statistical evaluation of virulence between strains can be easily achieved. The experimental design employs young gnotobiotic rats infected with S. mutans (day 19) and fed a purified diet (305) containing only 5% sucrose for test periods not exceeding 26 days. Rats infected with S. mutans 6715 develop rampant caries during this period, a level at the upper limits for evaluation of caries by the Keyes procedure. In addition, 35-day-old rats infected with this strain for 16 days had extensive lesions on all molar surfaces; however, their mean caries scores were two-fold lower than seen in 45-day-old animals. These results confirm previous findings that S. mutans 6715 causes dental caries in gnotobiotic rats (34, 35); however, these studies employed a caries-promoting diet which contained 56% sucrose, 11-fold more sucrose than is present in diet 305. In addition, these earlier studies employed significantly longer test periods. The high virulence of S. mutans 6715 observed in this study could be attributed to several factors. Our strain of S. mutans might be more virulent than other strains of 6715 previously tested or the Fischer gnotobiotic rat could be more susceptible to S. mutans pathogenesis. Although sucrose is considered essential for S. mutans adherence to tooth enamel and acid production, our results indicate that unusually high concentrations are necessary for development of severe carious lesions. Of interest was the observation that gnotobiotic rats infected with S. mutans 6715 and fed a sucrose-free purified diet (300) for 6 or 26 days remained colonized with the test organism. In this regard, previous studies have suggested that sucrose is not required for S. mutans colonization (3, 4, 15, 24). However, it appears to be a necessary dietary component required for maximum virulence of S. mutans in vivo. This was suggested by the experiment where infected rats were fed diet 300 for 26 days and the level of smooth surface lesions observed was sevenfold lower than occurred in infected animals fed diet 305 for the same length of time. That infected rats fed diet 300 for 26 days developed some lesions was not unexpected, since rats have alpha-amylases in saliva and the gut which can hydrolyze starch to glucose and maltose components (1, 26, 33). Earlier studies have demonstrated that rats are more susceptible to caries attack during the first few days after weaning and resistance increases with tooth maturation (25). In this regard, it appears that maximum sucroseinduced caries can be effected in 19- to 25-dayold gnotobiotic rats monoinfected with S. mu- 73

6 74 MICHALEK, McGHEE, AND NAVIA INFECT. IMMUN U) z04 I- "mm x10 z to : U)' 00 :0 I - I.- 10 z L6 IH_ n0 U ILw,T%f lh 20H_ AHT ENAMEL DENTIN ENAMEL DENTIN FIG. 3. Comparison of percentage of buccal (open bar) and proximal (shaded bar) lesions of S. mutans AHT (A), BHT (B), (C), and LM-7 (D) with S. mutans 6715 in 45-day-old gnotobiotic rats (Table 2, group D). tans and fed a caries-promoting diet. This was suggested from the experiments demonstrating that infected rats fed diet 305 from 25 to 35 days of age had mean caries scores which were not significantly different from the scores in 35-dayold animals fed diet 305 for 16 days. When the virulence of four strains of S. mutans (AHT, BHT, NCTC 10449, and LM-7) was compared with 6715, differences in the level of smooth surface caries were readily apparent. Although S. mutans AHT, BHT, NCTC 10449, and 6715 were highly virulent, animals infected with AHT had more buccal dentinal lesions than the other strains tested. On the other hand, animals infected with S. mutans 6715 had significantly more proximal lesions. This series of experiments suggests that different strains of A BHT 0-~~~~~~~~D LM -7 S. mutans preferentially attack smooth surfaces, as illustrated by S. mutans 6715, which caused greater proximal caries, whereas S. mutans AHT initiated severe buccal lesions. Of the five strains tested, S. mutans LM-7 was the least virulent, resulting in approximately twofold fewer smooth surface lesions than observed with S. mutans Although the strains tested corresponded to the standard S. mutans serotypes (2), much larger numbers of human isolates from each group must be tested before any correlation between group specificity and virulence can be made. ACKNOWLEDGMENTS We wish to thank Harold Fullmer, Tetsuo Shiota, and Annie Jo Narkates for their valuable suggestions and criti- B

7 VOL. 12, 1975 VIRULENCE OF S. MUTANS IN GNOTOBIOTIC RATS 75 cisms, and we gratefully acknowledge Jackie Morris for the typing of this manuscript. This investigation was supported by a grant from the John A. Hartford Foundation, Inc. and Public Health Service grants DE , DE , and DE from the National Institute of Dental Research. LITERATURE CITED 1. Borgstrom, B., A. Dahlquist, B. E. Gustafsson, G. Lundh, and J. Malmquist Trypsin, invertase and amylase content of feces of germfree rats. Proc. Soc. Exp. Biol. Med. 102: Bratthall, D Demonstration of five serological groups of streptococcal strains resembling Streptococcus mutans. Odontol. Revy 21: Carlsson, J Zooglea-forming streptococci, resembling Streptococcus sanguis, isolated from dental plaque in man. Odontol. Revy 16: Carlsson, J Presence of various types of nonhaemolytic streptococci in dental plaque and in other sites of the oral cavity in man. Odontol. Revy 18: Charlton, G., D. B. Fitzgerald. and P. H. 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