Dental Caries Induction in Experimental Animals by Clinical Strains of Streptococcus mutans Isolated from Japanese Children

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1 Microbiol. Immunol. Vol. 22 (6), , 1978 Dental Caries Induction in Experimental Animals by Clinical Strains of Streptococcus mutans Isolated from Japanese Children Shigeyuki HAMADA, Takashi OOSHIMA, Mitsuo TORII, Hideaki IMANISHI, Norio MASUDA, Shizuo SOBUE, and Shozo KOTANI Departments of Oral Microbiology, Pedodontics, and Operative Dentistry, Osaka University Dental School, Osaka (Received for publication, October 27, 1977) Abstract Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin-resistance were examined in specific pathogen-free Sprague-Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries-inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH- TR and FAlR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees. Several different species of streptococci have been isolated from human teeth and most of them are taxonomically identifiable, including Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitior (mitis) and some enterococcus (9). In 1960, the inoculation of S. mutans into the oral cavity of rodents was first shown to result in dental caries formation if the animals were fed a caries-inducing high sucrose diet for extended periods of time (8, 17). Epidemiological studies on humans indicated a positive correlation between the caries status and the prevalence of S. mutans in dental plaque or carious lesions (3, 13, 29). It is now widely accepted that S. mutans is highly effective in inducing experimental caries in a variety of animals such as hamsters, rats, gerbils and even monkeys (1, 5, 6, 10). Several investigators reported that bacteria other than S. mutans also 301

2 302 S. HAMADA ET AL induced caries in hamsters and rats under certain experimental conditions. However, cariogenicity was variable among the strains tested, even within the same bacterial species, and was to some extent a reflection on the animal test system. In preceding studies, we have isolated and serotyped hundreds of S. mutans strains from carious lesions or dental plaque of Japanese children (11, 24), but their cario- genicity was not determined. This study was undertaken to investigate the cariogenic potentials of the clinically isolated strains of S. mutans using animal model systems. In addition, some strains of oral streptococci and lactobacilli were tested for their cariogenicity using the same model systems. MATERIALS AND METHODS Bacterial strains. S. mutans strains of serotypes c, d, e, and f were employed in this study. These strains were isolated from carious lesions of Japanese children (11). Reference S. mutans strains BHTR (serotype b), FAlR (b) and OMZ176R (d) were also examined for cariogenic activity. Strains of S. sanguis and S. salivarius were isolated from dental plaque and saliva, respectively. Species designation of these strains was performed according to their colonial morphology on Mitis Salivarius agar plates (Difco), their fermentation patterns for various sugars, and other biochemical activities (11, 12). Table 1 shows various physiological properties of the streptococcal strains employed for caries testing. Lactobacillus acidophilus ATCC 4357 was obtained from Dr. Y. Kobayashi (Aichi Gakuin University School of Dentistry, Nagoya). The following strains of lactobacilli were kindly supplied by the Yakult Institute for Microbiological Research, Tokyo. Lactobacillus fermentum YIT82R is a substrain of strain ATCC 1492, isolated from human saliva. Lactobacillus casei YIT13OR and Lactobacillus helveticus YIT100R were isolated from human feces and fermented milk, respectively. All strains described above were labeled with resistance to streptomycin (1,500 per ml of culture medium) to facilitate recovery and identification of the implanted bacteria. The suffix R refers to streptomycin resistance. Animals and experimental design of caries testing. Specific pathogen-free Sprague- Dawley (SD) rats (15+1 days old) were obtained from the barrier-maintained colony of the Animal Center of Osaka University or Clea Japan, Inc., Osaka. They were derived from germfree ancestors and were barrier-maintained in a closed room until 15 days after birth. Then they were reared in the conventional manner. S. mutans could not be detected in their indigenous oral flora by our bacteriological examinations. Rat pups were randomly divided into appropriate groups. The general design of the caries test employed in the present study is presented in Fig. 1. Weanling male SD rats 15+1 days old were given ordinary powdered diet MF (Oriental Yeast Co., Osaka) and deionized water ad libitum. On the following three days of the experiment, the rats were given diet MF containing 0.4% tetracycline and drinking water containing penicillin G (4,000 units/ml) to suppress the indigenous flora of the rats. On the fourth day of the experiment, the rats were fed diet MF and water free of antibiotics. On the fifth day, the rats were infected with a heavy

3 CARIES INDUCTION BY S. MUTANS IN ANIMALS 303

4 304 S. HAMADA ET AL Fig. 1. Experimental design for testing cariogenicity of various bacteria. S. mutans strain MT615R and other bacterial species examined were inoculated according to procedure I. Strains of S. mutans, except MT615R, were implanted by procedure II. Swab samples from the oral cavity were taken at appropriate intervals after inoculation as shown in Tables 2, 3, and 4. cell suspension containing 1012 colony-forming-units (CFU) of the appropriate test strain using a pipette. They also received drinking water which contained 1010 CFU of fresh bacteria per ml of water. A control was swabbed with deionized water. At the same time, the diet was replaced with caries-inducing high sucrose diet #2000 (18). The inoculations were repeated as shown in Fig. 1. Diet #2000 was selected because of its extensive use by many research workers to produce smooth surface lesions as well as sulcal lesions (25). All animals were routinely checked to verify the establishment and persistence of the inoculated bacteria as well as for contamination, using Mitis Salivarius agar (Difco) for streptococci and SL agar (Difco) for lactobacilli, each containing 500 Đg per ml of streptomycin. The number of colonies recovered was recorded and the results were graded according to the description of Krasse (20). After an experimental period ranging from 85 to 122 days, the animals were sacrificed and the heads were defleshed by autoclaving for 5 min at 121 C. The method of caries assessment was essentially that of Keyes (15) in which the number of tooth surfaces and depths of lesions were evaluated. In some cases, the jaws were stained with erythrosin to evaluate the plaque index (26) before defleshing. To test whether the cariogenic potential of a strain of S. mutans depends on the animal species examined, golden hamsters and ICR mice were also employed for caries testing. The golden hamsters were purchased from a local dealer. ICR mice were obtained from Clea Japan, Inc. Preliminary examination demonstrated no S. mutans in the indigeneous oral flora of either animal species. RESULTS Recovery of the Implanted Streptococci The degree of recovery of three strains, representing different streptococcal species, from infected SD rats are tabulated in Tables 2, 3, and 4. S. mutans strain MT615R became easily established after the first inoculation series (Fig. 1, and

5 CARIES INDUCTION BY S. MUTANS IN ANIMALS 305 Table 2. Recovery of inoculated S. mutans MT615R from rats fed diet 2000 Degree of recovery was evaluated according to the description of Krasse (20). Inoculation of test bacterium was repeated when the infection was not proven to be established by bacteriological examination. The schedule of repeated inoculation is shown in Fig. 1. Table 3. Recovery of inoculated S. sanguis ST7R from rats fed diet 2000 cf. Footnote of Table 2. Table 4. Recovery of inoculated S. salivarius HT9R from rats fed diet 2000 cf. Footnote of Table 2. Table 2). Implanted streptococci persisted and proliferated on the teeth, and they were recovered in large numbers throughout the experimental period. S. sanguis strain ST7R was also established in the rat mouth but with some difficulties when compared with S. mutans. A few rats required repeated inoculations to be definitely infected (Table 3). Once established, however, the implanted streptococci per-

6 306 S. HAMADA ET AL Table 5. Caries induction in rats infected with various streptococci sisted and were recovered regularly by routine bacteriological examination. On the other hand, S. salivarius strain HT9R was recovered sporadically even after repeated inoculations (Table 4). Cariogenic Activity of the Streptococcal Strains in Sprague-Dawley Rats All the clinical isolates (7 strains) of S. mutans, belonging to serotypes c, d, e, and f, developed heavy gelatinous plaque on the molar teeth and induced dental caries in SD rats which was statistically significant when compared with noninfected control rats (Table 5). Three reference strains, BHTR and FAlR (both serotype b) and OMZ176R.(d), were also cariogenic; but the cariogenic potential of the two reference type b strains was rather weak compared with those of the clinical isolates of S. mutans. Both strains, a clinical isolate MT615R and a reference OMZ176R of type d S. mutans induced marked smooth surface caries on the buccal and lingual surfaces of the teeth (Fig. 2). On the other hand, type c, e, or f strains preferably induced caries originating from pits and fissures (Fig. 3) rather than smooth surface caries. The cariogenicity of some strains of S. sanguis or S. salivarius was found to be very weak or absent. Among these strains, only S. sanguis strain ST7R induced small but definite caries. Three negative control groups (eight rats each), which were noninfected and maintained under the same conditions as the infected test groups, did not develop

7 CARIES INDUCTION BY S. MUTANS IN ANIMALS Fig. 2. Smooth surface caries in the mandibular rat inoculated with S. unarms NI16L5R (serotype Fig. 3. Fissure caries in the molar S. mama MT6R (type r). teeth molar d). of rats inoculated 307 teeth of with any significant carious lesions (Fig. 4a, b). It was noted that increases in body weight of the animals were essentially similar among the test and control groups. Implantation and Cariogenicity of Lactobacilli in Sprague-Dawley Rats Four strains belonging to Liermentum, L. acidophilus, L. casei and L. helvelicuswere

8 S.HAMADA 308 ET AL a b Fig. 4a, b. The carics-inducing Fable 6. molar teeth diet #2000. Caries induction of a noninkrted in rats infected control with rat lactobacilli fed a

9 CARIES Table 7. Caries INDUCTION induction in hamsters, BY S. MUTANS rats, and mice Fig. 5. Heavy accumulation of gelatinous erythrosin. Massive underlying cavitation molars was observed after removing the IN ANIMALS infected with 309 S. matans MT6R plaque stained with of second and third plaque (d. Fig. 6a). investigated concerning their cariogenicity in SD rats. As with S. salivarius, all lactobacilli strains required repeated inoculations to be recovered in significant numbers and to persist during the experimental period (data not shown). None of these strains developed definite carious lesions (Table 6). Caries Induction in Various Animal Species with a Strain of S. mutans S. mutans strain MT6R (a serotype c clinical isolate) was inoculated into golden hamsters and ICR mice in addition to Sprague-Dawley rats, to compare the caries susceptibility of these animal species (Table 7). This strain produced marked plaque accumulations on the molar teeth (Fig. 5) and developed more extensive smooth surface caries in all golden hamsters than those in rats (Fig. 6a, b). The inoculated strain could also be implanted in all ICR mice and caries developed (Fig. 7a, b).

10 310 S. HAMADA ET AI a b Fig. 6. Extensive carious lesions in the molar teeth of a golden hamster infected with S. mulans MT6R (a). There were no lesions in the control hamster (b). However, caries scores in the mice were far smaller than those in hamsters and rats, and significant individual variations of caries scores were observed in mice (Table 7). DISCUSSION Scvcralapproachcsllavcbccndevisedfbraninlalcariesstudicswitildifferent

11 CARIES INDUCTION BY S. MUTANS 311 IN ANIMALS a b Fig. 7. Carious lesions in the molar with S. mutaii., NITGR (a). Noninlected of caries (b). teeth of control ICR mice mouse infected were found free infection statuses such as germ-freeignotobiotic, antibiotic depressed (relative gnotobiotic) and conventional animals (19, 25). It has been shown that experimental animals, which normally do not develop dental caries, will develop caries when infected with one of the cariogenic streptococci and fed a sucrose-containing diet (16, 22). In the present study, specific pathogen free (SPF) Sprague-Dawley rats

12 312 S. HAMADA ET AL were purchased and reared in the conventional state for caries testing. They developed no definite dental caries even when fed a high sucrose diet, indicating that no cariogenic bacteria established themselves in the oral microflora. Therefore, cariogenicity of a bacterium can be assessed by superinfecting it into rats and feeding them with high sucrose diet #2000. The oral flora were depressed by use of antibiotics prior to inoculation of the test strain because under some conditions the indigenous flora may compete with newly inoculated bacteria (22). Using this animal model system, all the clinical isolates of S. mutans tested were shown to be easily established in the mouths of experimental animals and were significantly cariogenic. These strains represent each serotype of S. mutans which has been found in Japanese children (11). Among these strains, it should be noted that serotype d strains, MT615R and OMZ176R, preferentially developed smooth surface caries as well as caries of fissure origin. On the other hand, strains of the other serotypes induced carious lesions primarily confined to the sulcal area of the occlusal surface. It was of interest that the reference strains, BHTR and FA1R of type b, were found to have significantly diminished cariogenic activity. The original strains were reported to induce severe caries in rats (8, 33). The attenuation of cariorgenicity in these strains was probably due to serial passages of the strains in the laboratories or due to differences in the animals employed in the experiments. The failure or difficulty of S. salivarius strains to become established in the mouth of the rats was noted. On the other hand, S. sanguis strains could be implanted in the rat mouth. These results were of interest because it had been found that most strains of S. salivarius can be implanted in the hamster mouth whereas those of S. sanguis cannot (14, 21). We failed to demonstrate strong cariogenic activity of S. sanguis strains which were established in the mouths of conventional rats. In this connection, it had been reported that severe dental caries was induced in some cases in the gnotobiotic or relatively gnotobiotic rats infected with S. sanguis strains (10). The comparative study of caries development in three animal species, fed the same caries-inducing diet and infected with a clinically isolated S. mutans strain MT6R (serotype c), suggests that there are substantial species differences relevant to caries induction. Rats and hamsters were significantly susceptible to the cariogenic challenge of human strain MT6R. It is of interest to note that hamster caries was primarily of smooth surface origin, probably because of the anatomical features of the hamster molars (25). Mice were far less susceptible to cariogenic attack by the same strain, and individual variations of caries scores in mice were significant. Similar findings were reported by StrAlfors et al (30), while Sharawy and Socransky (28) failed to implant S. mutans and induce dental caries in mice. These findings suggested that mice are not suitable as a caries model despite the suggestion by Navia (25) that their hardiness, small body size, economic maintenance and genetic homogeneity should qualify mice as useful in dental caries research. Significant variations in caries scores observed in all animal species might be attributed in part to genetical heterogeneity of the animals employed, the difference of animal age (15 } days as the starting age), and the variation of oral microflora

13 CARIES INDUCTION BY S. MUTANS IN ANIMALS 313 of conventional animals. It has been shown that the age of the tooth is critically important for susceptibility to colonization by S. mutans and the resultant formation of caries (22, 32). It has been shown that in humans, high counts of salivary lactobacilli can be correlated with prevalance of dental caries (2). However, very few strains of lactobacilli belonging to L. casei and L. acidophilus have been reported to be cariogenic in gnotobiotic rats (7, 27). The present study also suggests that several lactobacilli, including L. casei, retain no cariogenic activity. It should be mentioned here that among the Lactobacillus species, L. casei is known to be the most common species in carious dentin in humans (23, 31). L. casei is also reported to be one of the most aciduric among the lactobacilli (4). In conclusion, the present results add further evidence to the view that S. mutans have some important properties responsible for caries induction which other species of streptococci and lactobacilli lack. This property of S. mutans is one of the most important research subjects which still remain to be elucidated in future studies. We thank Dr. Rosemary Linzer for correcting the English version of the manuscript. This research was supported in part by a grant (257464) from the Ministry of Education, Science and Culture of Japan. REFERENCES 1) Bowen, W.H The induction of rampant dental caries in monkeys ( Macaca irus ). Caries Res. 3: ) Davis, G.N., King, R.M., and Collins, A.A The relationship between lactobacillus counts, Snyder tests and the subsequent incidence of dental caries. Arch. Oral Biol. 1: ) De Stoppelaar, J.D., Van Houte, J., and Baker Dirks, O The relationship between extracellular polysaccharide-producing streptococci and smooth surface caries in 13-year-old children. Caries Res. 3: ) Featherstone, J.L The oral lactobacilli of central Australian aborigines. II. Differentiation of species. Aust. Dent. J. 4: ) Fitzgerald, R.J Dental caries research in gnotobiotic animals. Caries Res. 2: ) Fitzgerald, D.B., and Fitzgerald, R.J Induction of dental caries in gerbils. Arch. Oral Biol. 11: ) Fitzgerald, R. J., Jordan, H.V., and Archard, H.O Dental caries in gnotobiotic rats infected with a variety of Lactobacillus acidophilus. Arch. Oral Biol. 11: ) Fitzgerald, R.J., and Keyes, P.H Demonstration of the etiologic role of streptococci in experimental caries in the hamster. J. Am. Dent. Assoc. 61: ) Gibbons, R.J., and Van Houte, J Dental caries. Ann. Rev. Med. 26: ) Guggenheim, B Streptococci of dental plaques. Caries Res. 2: ) Hamada, S., Masuda, N., Ooshima, T., Sobue, S., and Kotani, S Epidemiological survey of Streptococcus mutans among Japanese children. Identification and serological typing of the isolated strains. Japan. J. Microbiol. 20: ) Hamada, S., Masuda, N., and Torii, M Isolation and identification of cariogenic streptococci. Nippon Dent. Rev. 415: ) Ikeda, T., Sandham, H. J., and Bradley, E.L., Jr Changes in Streptococcus mutans and lactobacilli in plaque in relation to the initiation of dental caries in Negro children. Arch. Oral Biol. 18: ) Jordan, H.V., and Van Houte, J The hamster as an experimental model for odontopathic infections. Prog. Exp. Tumor Res. 16: ) Keyes, P.H A method of recording and scoring gross carious lesions in the molar teeth of Syrian hamster. J. Dent. Res. 23:

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